Liver-specific transgenic expression of cholesteryl ester hydrolase reduces atherosclerosis in Ldlr−/− mice

The liver plays a central role in the final elimination of cholesterol from the body either as bile acids or as free cholesterol (FC), and lipoprotein-derived cholesterol is the major source of total biliary cholesterol. HDL is the major lipoprotein responsible for removal and transport of cholesterol, mainly as cholesteryl esters (CEs), from the peripheral tissues to the liver. While HDL-FC is rapidly secreted into bile, the fate of HDL-CE remains unclear. We have earlier demonstrated the role of human CE hydrolase (CEH, CES1) in hepatic hydrolysis of HDL-CE and increasing bile acid synthesis, a process dependent on scavenger receptor BI expression. In the present study, we examined the hypothesis that by enhancing the elimination of HDL-CE into bile/feces, liver-specific transgenic expression of CEH will be anti-atherogenic. Increased CEH expression in the liver significantly increased the flux of HDL-CE to bile acids. In the LDLR−/− background, this enhanced elimination of cholesterol led to attenuation of diet-induced atherosclerosis with a consistent increase in fecal sterol secretion primarily as bile acids. Taken together with the observed reduction in atherosclerosis by increasing macrophage CEH-mediated cholesterol efflux, these studies establish CEH as an important regulator in enhancing cholesterol elimination and also as an anti-atherogenic target. The liver plays a central role in the final elimination of cholesterol from the body either as bile acids or as free cholesterol (FC), and lipoprotein-derived cholesterol is the major source of total biliary cholesterol. HDL is the major lipoprotein responsible for removal and transport of cholesterol, mainly as cholesteryl esters (CEs), from the peripheral tissues to the liver. While HDL-FC is rapidly secreted into bile, the fate of HDL-CE remains unclear. We have earlier demonstrated the role of human CE hydrolase (CEH, CES1) in hepatic hydrolysis of HDL-CE and increasing bile acid synthesis, a process dependent on scavenger receptor BI expression. In the present study, we examined the hypothesis that by enhancing the elimination of HDL-CE into bile/feces, liver-specific transgenic expression of CEH will be anti-atherogenic. Increased CEH expression in the liver significantly increased the flux of HDL-CE to bile acids. In the LDLR−/− background, this enhanced elimination of cholesterol led to attenuation of diet-induced atherosclerosis with a consistent increase in fecal sterol secretion primarily as bile acids. Taken together with the observed reduction in atherosclerosis by increasing macrophage CEH-mediated cholesterol efflux, these studies establish CEH as an important regulator in enhancing cholesterol elimination and also as an anti-atherogenic target. Homeostatic balance between dietary intake, endogenous synthesis, and fecal elimination of cholesterol is fessential to prevent pathological accumulation of cholesterol in macrophage foam cells that leads to the development of atherosclerosis. Unlike other macromolecules such as carbohydrates, proteins, or nucleic acids, once synthesized the steroid nucleus of cholesterol cannot be degraded within the human body and excess cholesterol can only be removed following biotransformation to more water soluble bile acids or as free cholesterol (FC) solubilized in bile acids. Because bile acid synthesis and secretion of cholesterol into the bile is largely restricted to the liver, the liver plays a central role in regulating the elimination of cholesterol from the body. Furthermore, as only a small portion (5–20*) of biliary cholesterol is derived from de novo synthesis (1Robins S.J. Fasulo J.M. Lessard D. Patton G.M. Hepatic cholesterol synthesis and the secretion of newly synthesized cholesterol in bile.Biochem. J. 1993; 289: 41-44Crossref PubMed Scopus (18) Google Scholar, 2Empen K. Lange K. Stange E.F. Newly synthesized cholesterol in human bile and plasma: quantification by mass isotopomer distribution analysis.Am. J. Physiol. Gastrointest. Liver Physiol. 1997; 272: G367-G373Crossref PubMed Google Scholar) and the bulk is supplied by the hepatic uptake of lipoproteins (3Schwartz C.C. Berman M. Vlahcevic Z.R. Halloran L.G. Gregory D.H. Swell L. Multicompartmental analysis of cholesterol metabolism in man. Characterization of the hepatic bile acid and biliary cholesterol precursor sites.J. Clin. Invest. 1978; 61: 408-423Crossref PubMed Scopus (133) Google Scholar, 4Schwartz C.C. Halloran L.G. Vlahcevic Z.R. Gregory D.H. Swell L. Preferential utilization of free cholesterol from high density lipoproteins for biliary cholesterol secretion in man.Science. 1978; 200: 62-64Crossref PubMed Scopus (217) Google Scholar), the liver also plays a key role in the flux of cholesterol returning to the liver from the peripheral tissues via lipoproteins. Chylomicrons carrying the dietary cholesterol return to the liver as remnants, and following delivery of associated TGs and cholesterol to the peripheral tissues, liver-derived VLDLs return to the liver as LDLs (5Gotto Jr, A.M. Interrelationship of triglycerides with lipoproteins and high-density lipoproteins.Am. J. Cardiol. 1990; 66: 20A-23AAbstract Full Text PDF PubMed Scopus (35) Google Scholar). This hepatic uptake of remnant or LDL-associated cholesterol is thought to regulate hepatic cholesterol synthesis or VLDL secretion (6Brown M.S.J.L.G. A receptor-mediated pathway for cholesterol homeostasis.Science. 1986; 232: 34-47Crossref PubMed Scopus (4350) Google Scholar), and recently Sniderman et al. (7Sniderman A.D. Qi Y. Ma C.L. Wang R.H. Naples M. Baker C. Zhang J. Adeli K. Kiss R.S. Hepatic cholesterol homeostasis: is the low-density lipoprotein pathway a regulatory or a shunt pathway?.Arterioscler. Thromb. Vasc. Biol. 2013; 33: 2481-2490Crossref PubMed Scopus (24) Google Scholar) have demonstrated that while chylomicron remnant-associated cholesterol enters the regulatory pool and modulates hepatic de novo synthesis, LDL cholesterol is resecreted as VLDL. In contrast to LDL, HDL removes cholesterol from peripheral tissues, including artery wall-associated macrophage foam cells, delivers it to the liver and represents the major mechanism for the flux of cholesterol from the peripheral tissues to the liver by the process of reverse cholesterol transport (RCT). Because cholesterol is carried as cholesteryl esters (CEs) in all lipoproteins, intracellular hydrolysis of CEs is obligatory for the release of FC within the hepatocyte. While the remnants and LDLs are taken up by the endocytic pathway and the associated CEs are hydrolyzed within the acidic lysosomal compartment by acid CE hydrolase (CEH), CEs associated with HDLs enter the hepatocyte by selective uptake pathway via scavenger receptor BI (SR-BI) and hydrolysis of HDL-CEs is extra lysosomal and catalyzed by a neutral CEH (8Shimada A. Tamai T. Oida K. Takahashi S. Suzuki J. Nakai T. Miyabo S. Increase in neutral cholesteryl ester hydrolase activity produced by extralysosomal hydrolysis of high-density lipoprotein cholesteryl esters in rat hepatoma cells (H-35).Biochim. Biophys. Acta. 1994; 1215: 126-132Crossref PubMed Scopus (26) Google Scholar). We purified (9Ghosh S. Grogan W.M. Rapid three-step purification of a hepatic neutral cholesteryl ester hydrolase which is not the pancreatic enzyme.Lipids. 1991; 26: 793-798Crossref PubMed Scopus (28) Google Scholar), characterized, and cloned rat liver neutral CEH (10Ghosh S. Mallonee D.H. Hylemon P.B. Grogan W.M. Molecular cloning and expression of rat hepatic neutral cholesteryl ester hydrolase.Biochim. Biophys. Acta. 1995; 1259: 305-312Crossref PubMed Scopus (56) Google Scholar), a member of the carboxylesterase family, and established its role in hepatic cholesterol homeostasis (11Ghosh S. Natarajan R. Pandak W.M. Hylemon P.B. Grogan W.M. Regulation of hepatic neutral cholesteryl ester hydrolase by hormones and changes in cholesterol flux.Am. J. Physiol. 1998; 274: G662-G668PubMed Google Scholar). Recently, we also cloned and characterized human liver CEH and demonstrated that transient over-expression of CEH increased bile acid synthesis and secretion from primary human hepatocytes (12Zhao B. Natarajan R. Ghosh S. Human liver cholesteryl ester hydrolase: cloning, molecular characterization, and role in cellular cholesterol homeostasis.Physiol. Genomics. 2005; 23: 304-310Crossref PubMed Scopus (50) Google Scholar). Furthermore, adenovirus-mediated over-expression of this enzyme enhanced cholesterol elimination by increasing the flux of cholesterol from macrophages to feces (in vivo RCT) (13Zhao B. Song J. Ghosh S. Hepatic overexpression of cholesteryl ester hydrolase enhances cholesterol elimination and in vivo reverse cholesterol transport.J. Lipid Res. 2008; 49: 2212-2217Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). More importantly, SR-BI deficiency completely abolished CEH-mediated increase in in vivo RCT, demonstrating the requirement of functional SR-BI for CEH to channel HDL-derived CEs to bile and feces (13Zhao B. Song J. Ghosh S. Hepatic overexpression of cholesteryl ester hydrolase enhances cholesterol elimination and in vivo reverse cholesterol transport.J. Lipid Res. 2008; 49: 2212-2217Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Direct association of CEH with SR-BI delivered HDL-CEs, and conversion of HDL-CEs to bile acids further confirmed the role of hepatic CEH in metabolizing HDL-CEs and making the FC available for bile acid synthesis (14Yuan Q. Bie J. Wang J. Ghosh S.S. Ghosh S. Cooperation between hepatic cholesteryl ester hydrolase and scavenger receptor BI for hydrolysis of HDL-CE.J. Lipid Res. 2013; 54: 3078-3084Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Consistently, liver-specific deletion of Ces3, the murine homolog of human CEH (hCEH), led to a significant decrease in bile acid secretion and fecal elimination of bile acids resulting in an increase in diet-induced atherosclerosis in LDL receptor (LDLR)−/− mice (15Bie J. Wang J. Marqueen K.E. Osborne R. Kakiyama G. Korzun W. Ghosh S.S. Ghosh S. Liver-specific cholesteryl ester hydrolase deficiency attenuates sterol elimination in the feces and increases atherosclerosis in ldlr-/- mice.Arterioscler. Thromb. Vasc. Biol. 2013; 33: 1795-1802Crossref PubMed Scopus (24) Google Scholar). In the present study we sought to further establish the anti-atherogenic role of hepatic CE hydrolysis by developing liver-specific hCEH transgenic mice. Hepatic over-expression of CEH did not affect hepatic cholesterol homeostasis but led to an increase in the flux of HDL-CEs to bile acids. The data presented here also demonstrates that by increasing fecal elimination of cholesterol as bile acids, liver-specific transgenic expression of CEH leads to attenuation of diet-induced atherosclerosis in LDLR−/− mice. The plasmid pLIV.11, containing human apoE promoter as well as hepatic control region and all the necessary elements for chimeric transgene construction (poly linker region and heterologous intron), was used (16Simonet W.S. Bucay N. Lauer S.J. Taylor J.M. A far-downstream hepatocyte-specific control region directs expression of the linked human apolipoprotein E and C–I genes in transgenic mice.J. Biol. Chem. 1993; 268: 8221-8229Abstract Full Text PDF PubMed Google Scholar). Full-length hCEH cDNA (∼2 kb) with an inframe 3′-c-myc epitope was cloned into the MunI and MluI sites in the poly linker region (supplementary Fig. I), and the sequence of the chimeric transgene was confirmed by sequencing. The chimeric transgene was excised by digestion with SalI and SpeI, purified by agarose gel electrophoresis, and injected into the pronuclei of fertilized mouse eggs obtained from super-ovulated female mice (Balb-c/C57BL/6 hybrids). The injected eggs were surgically transferred to oviducts of surrogate females. Presence of hCEH transgene was confirmed either by PCR amplification of a 682 bp product using hCEH specific primers (133–155 bp and 814–792 bp) using mouse tail genomic DNA as a template or by Southern blot analysis using full-length hCEH as a probe to identify the ∼1,265 bp integrated DNA. The founder mice in the Balb-c/C57BL/6 hybrid background were backcrossed into the C57BL/6 background for 10 generations before experimentation and were labeled as liver-specific hCEH transgenic (LCEH2) mice. For evaluation of atherosclerosis, LCEH2 mice were crossed into the LDLR−/− background. To generate macrophage- and liver-specific double transgenics in the LDLR−/− background, LDLR−/−LCEH2 mice were crossed with LDLR−/−CEHTg mice generated in our laboratory earlier (17Zhao B. Song J. Chow W.N. St Clair R.W. Rudel L.L. Ghosh S. Macrophage-specific transgenic expression of cholesteryl ester hydrolase significantly reduces atherosclerosis and lesion necrosis in Ldlr mice.J. Clin. Invest. 2007; 117: 2983-2992Crossref PubMed Scopus (109) Google Scholar). Male and female littermates were included in the study at 10 weeks of age. The total number of animals in each group was, therefore, determined by the availability of the correct genotype and gender within a litter. For assessment of atherosclerosis, mice were fed a Western-type high-fat/high-cholesterol diet (TD88137, Harlan Teklad), which contained 21* fat, 0.15* cholesterol, and 19.5* casein by weight with no sodium cholate for 16 weeks. All procedures were approved by the Virginia Commonweath University Institutional Animal Care and Use Committee. Littermates were used for all experiments. Tissues were harvested from WT (C57BL/6) and LCEH2 mice and total RNA was extracted using an RNeasy kit (Qiagen). hCEH mRNA expression was determined by real time PCR using optimized TaqMan gene expression assay (Hs00275607_m1), and mRNA copy number was determined using a standard curve as described earlier (12Zhao B. Natarajan R. Ghosh S. Human liver cholesteryl ester hydrolase: cloning, molecular characterization, and role in cellular cholesterol homeostasis.Physiol. Genomics. 2005; 23: 304-310Crossref PubMed Scopus (50) Google Scholar). To determine the hCEH protein expression, total protein extracts prepared from different tissues were analyzed by Western blot analysis using anti-c-myc antibody to specifically identify the c-myc tag on transgenic hCEH and species-specific fluorescently labeled secondary antibodies. Positive immune-reactivity was detected by scanning in the appropriate channels with an Odyssey infrared imaging system (LI-COR). Primary hepatocytes were prepared, and cytoplasm as well as endoplasmic reticulum (ER) were fractionated by differential centrifugation. The presence of hCEH was detected by Western blot analysis as described above. CE hydrolytic activity was determined using a micellar substrate as described earlier (18Zhao B. Bie J. Wang J. Marqueen S.A. Ghosh S. Identification of a novel intracellular cholesteryl ester hydrolase (carboxylesterase 3) in human macrophages: compensatory increase in its expression after carboxylesterase 1 silencing.Am. J. Physiol. Cell Physiol. 2012; 303: C427-C435Crossref PubMed Scopus (20) Google Scholar). Livers were harvested from WT and LCEH2 mice and precision cut liver slices were incubated with [3H]oleate for 3 h (19Bie J. Zhao B. Marqueen K.E. Wang J. Szomju B. Ghosh S. Macrophage-specific transgenic expression of cholesteryl ester hydrolase attenuates hepatic lipid accumulation and also improves glucose tolerance in ob/ob mice.Am. J. Physiol. Endocrinol. Metab. 2012; 302: E1283-E1291Crossref PubMed Scopus (13) Google Scholar). Following three washes in PBS, total lipids were extracted and neutral lipids were separated by TLC using hexane:diethyl ether:acetic acid::90:10:1 (v/v). Spots corresponding to TG were marked, silica gel scraped, and associated radioactivity determined by liquid scintillation counting. To determine the rate of TG secretion in vivo, mice were fasted overnight and a baseline blood sample was collected via the tail vein. Mice were subsequently injected with tyloxapol (Sigma-Aldrich) at a concentration of 500 mg/kg body weight to inhibit lipoprotein lipase. Blood samples were subsequently collected at 1, 2, and 3 h postinjection and plasma TG levels were determined (L-Type TG-M kit, Wako Diagnostics). TG production rates were calculated as described (20Shachter N.S. Hayek T. Leff T. Smith J.D. Rosenberg D.W. Walsh A. Ramakrishnan R. Goldberg I.J. Ginsberg H.N. Breslow J.L. Overexpression of apolipoprotein CII causes hypertriglyceridemia in transgenic mice.J. Clin. Invest. 1994; 93: 1683-1690Crossref PubMed Scopus (119) Google Scholar). A modified Column Lipoprotein Profile method was used. Whole plasma aliquots frozen and stored at −80°C were thawed at 4°C. Total plasma cholesterol (TPC) concentration was determined by a micro enzymatic method as described earlier (17Zhao B. Song J. Chow W.N. St Clair R.W. Rudel L.L. Ghosh S. Macrophage-specific transgenic expression of cholesteryl ester hydrolase significantly reduces atherosclerosis and lesion necrosis in Ldlr mice.J. Clin. Invest. 2007; 117: 2983-2992Crossref PubMed Scopus (109) Google Scholar). In brief, approximately 20 ¼g of cholesterol was injected onto a fast-protein liquid chromatography system (Superose 6 HR 10/30 column, Amersham Biosciences) with online mixing of the column effluent with enzymatic reagent (Cholesterol Liquid Stable, Thermo Electron) for acquiring lipoprotein cholesterol profiles. The data were acquired on a personal computer running ChromPerfect Spirit chromatography software (Justice Software). The system was optimized so that the area under the profiles was proportional to the cholesterol mass. Area percent in each lipoprotein fraction, VLDL, LDL, and HDL, was applied to TPC to calculate cholesterol concentration in the lipoprotein fractions. About 100 mg of fresh liver tissue was homogenized in PBS and total lipids were extracted and amount of total cholesterol (TC), CEs, and TGs were determined, as described before, and normalized to wet weight. For histological analyses, liver tissue was fixed in 10* buffered formalin and paraffin embedded. Five micron sections were stained with hematoxylin and eosin and images were acquired using a Zeiss inverted microscope fitted with a digital camera as described earlier (19Bie J. Zhao B. Marqueen K.E. Wang J. Szomju B. Ghosh S. Macrophage-specific transgenic expression of cholesteryl ester hydrolase attenuates hepatic lipid accumulation and also improves glucose tolerance in ob/ob mice.Am. J. Physiol. Endocrinol. Metab. 2012; 302: E1283-E1291Crossref PubMed Scopus (13) Google Scholar). Ten-week-old LDLR−/− and LDLR−/−LCEH2 littermates were fed a Western diet for 16 weeks. After an overnight fast, a single bolus of glucose (2 mg/g body weight) was given intraperitoneally. Blood glucose levels were determined by commercially available glucometer using tail vein blood at 0, 15, 30, 60, 120, and 180 min. The aorta was dissected from the heart to the iliac bifurcation, cleaned of any surrounding tissue, opened longitudinally, pinned on black wax, and fixed for 24 h in 10* buffered formalin. The fixed aortas were imaged on a black background using a Canon digital camera fitted with a 60 mm f/2.8 Macro lens. Total area and the area occupied by the lesions in the aortic arch and total aorta were determined using AxioVision™ image analysis software. The person quantifying the area occupied by lesions was blinded to the identity of the images. Purified human HDL was purchased from Intracel and labeled with [3H]cholesteryl oleate using recombinant CETP as described earlier (15Bie J. Wang J. Marqueen K.E. Osborne R. Kakiyama G. Korzun W. Ghosh S.S. Ghosh S. Liver-specific cholesteryl ester hydrolase deficiency attenuates sterol elimination in the feces and increases atherosclerosis in ldlr-/- mice.Arterioscler. Thromb. Vasc. Biol. 2013; 33: 1795-1802Crossref PubMed Scopus (24) Google Scholar). Mice maintained on chow diet were injected (iv) with labeled HDL and transferred to metabolic cages. At 48 h, mice were euthanized and gall bladder bile was collected. Radioactivity associated with biliary cholesterol as well as bile acids was determined as described (17Zhao B. Song J. Chow W.N. St Clair R.W. Rudel L.L. Ghosh S. Macrophage-specific transgenic expression of cholesteryl ester hydrolase significantly reduces atherosclerosis and lesion necrosis in Ldlr mice.J. Clin. Invest. 2007; 117: 2983-2992Crossref PubMed Scopus (109) Google Scholar). TC was extracted from the dried feces using chloroform-methanol (2:1, v/v). After evaporation under nitrogen, the extracts were solubilized in 2-propanol containing 10* Triton X-100 TC estimated by enzymatic assay using a Wako® Cholesterol E test kit. Bile acids extracted from dried feces were derivatized and the resulting phenacyl esters were separated using reverse phase HPLC (15Bie J. Wang J. Marqueen K.E. Osborne R. Kakiyama G. Korzun W. Ghosh S.S. Ghosh S. Liver-specific cholesteryl ester hydrolase deficiency attenuates sterol elimination in the feces and increases atherosclerosis in ldlr-/- mice.Arterioscler. Thromb. Vasc. Biol. 2013; 33: 1795-1802Crossref PubMed Scopus (24) Google Scholar). Total fecal bile acids and cholesterol were normalized to the dry weight of the feces and data presented as micromoles per gram. Total RNA was extracted using an RNeasy kit (Qiagen). cDNA was synthesized using a high capacity cDNA reverse transcription kit (Applied Biosystems). Real time PCR was performed on a Stratagene Mx3000P machine, using TaqMan Universal PCR Master Mix and optimized probe and primer sets from Applied Biosystems. The following optimized probes were used: Ces3, Mm00474816_m1; Ces1, Mm00491334_m1; Ces5, Mm00555211_m1; ES22, Mm00504914_m1; ES1, Mm00468347_m1; ES31, Mm00519905_m1; HSL, Mm00495359_m1; TG hydrolase (Tgh)2, Mm00523518_m1; Kiaa, Mm00626772_m1; BSEP, Mm00445168_m1; Abcg5, Mm00446249_m1; Abcg8, Mm00445970_m1; MDR2, Mm00435630_m1; HMGCR, Mm01282501_m1; ACAT-2, Mm00448823_m1; Ldlr, Mm00440169_m1; CYP7A1, Mm00484152_m1; and liver X receptor (LXR), Mm00437265_g1. Data were analyzed by two-way ANOVA using GraphPad Prizm followed by Bonferroni post hoc tests to determine genotype and gender interactions, if any, as well as the significance of genotype and gender effects. Table 1 summarizes the results of these analyses including the actual P values. The differences were considered significant with P < 0.05 and are indicated in all figure legends.TABLE 1Two-way ANOVA analysesParameterGenotype/Gender InteractionGenotype EffectsGender EffectsTPCP = 0.002P = 0.01P = 0.0002VLDLNSP = 0.003NSLDLP = 0.002P = 0.09P = 0.0002HDLNSP = 0.02P = 0.05Arch lesion area (LDLR−/− vs. LDLR−/−LCEH2)NSP < 0.0001NSTotal lesion area (LDLR−/− vs. LDLR−/−LCEH2)NSP = 0.002NSArch lesion area (LDLR−/− vs. MLCL)NSP = 0.0004NSTotal lesion area (LDLR−/− vs. MLCL)NSP < 0.0001NSFecal BA (LDLR−/− vs. LDLR−/−LCEH2NSP = 0.002P = 0.001Fecal cholesterol (LDLR−/− vs. LDLR−/−LCEH2)NSNSP = 0.001Two-way ANOVA analyses were performed for the indicated parameters and significant differences due to genotype or gender as well as genotype/gender interactions are shown. BA, bile acid. Open table in a new tab Two-way ANOVA analyses were performed for the indicated parameters and significant differences due to genotype or gender as well as genotype/gender interactions are shown. BA, bile acid. Liver-specific hCEH (gene symbol CES1) transgenic mice were developed and backcrossed into the C57BL/6 background. Total RNA from multiple tissues was used to determine the expression of hCEH. Consistent with the expression driven by the apoE promoter and the hepatocyte control region, liver-specific expression of hCEH mRNA was seen in LCEH2 mice (Fig. 1A). Furthermore, high expression of c-myc tagged hCEH protein was detected in total protein extracts from the liver, confirming liver-specific expression (Fig. 1B). The minor immune-reactive protein seen in kidney and lung extracts and the detection of a low copy number of hCEH in other tissues is likely due to the nonspecific proximal enhancer element in the apoE promoter as described by Simonet et al. (16Simonet W.S. Bucay N. Lauer S.J. Taylor J.M. A far-downstream hepatocyte-specific control region directs expression of the linked human apolipoprotein E and C–I genes in transgenic mice.J. Biol. Chem. 1993; 268: 8221-8229Abstract Full Text PDF PubMed Google Scholar). Sub-cellular distribution of hCEH and CEH activity was determined in primary hepatocytes. The c-myc tagged CEH protein was associated with cytoplasm as well as ER; protein disulfide isomerase and GAPDH were used as markers for ER and cytoplasm, respectively (Fig. 1C). CEH-specific activity was increased by ∼3.5-fold in the cytoplasm and ∼2.6-fold in the ER of LCEH2 mice. However, transgenic CEH over-expression did not affect the relative distribution of total CEH activity; >85–90* of total activity was associated with cytoplasm, consistent with the cytoplasmic localization of CEH. Two independent transgenic lines of mice (LCEH1 and LCEH2) were developed with similar liver-specific expression and the LCEH2 line was chosen for all further studies. Human CES1 belongs to the carboxylesterase family and Ces3 is its murine homolog. To evaluate the effects, if any, on the expression of Ces3 and other members of the carboxylesterase family by transgenic expression of hCEH, expression of the indicated carboxylesterases was also monitored. No significant differences were noted in the expression of other genes (supplementary Fig. II) in mice on either a chow or a high-fat/high-cholesterol-containing Western diet. To determine the potential effects of transgenic expression of hCEH in affecting genes involved in maintaining hepatic cholesterol homeostasis, expression of HMGCR, ACAT-2, Ldlr, and CYP7A1 was assessed. While a nonsignificant increase in CYP7A1 and significant increase in the expression of SR-BI (P = 0.02 for chow-fed mice, P = 0.04 for Western diet-fed mice) was observed, there was no change in the expression of other genes (supplementary Fig. III). Expression of LXR, a gene that plays an important role in hepatic lipogenesis, also remained unaltered (1.00 ± 0.02 in WT mice vs. 1.05 ± 0.11 in LCEH2 mice). Because FC released as a result of CEH-mediated CE hydrolysis can either be directly secreted into bile via the FC transporter AbcG5/G8 or converted into bile acids before secretion via bile acid transporter (BSEP), expression of these transporters was evaluated. Ces3 deficiency did not affect the expression of these transporters (supplementary Fig. IV). There was also no change in the expression of phospholipid transporter MDR2. In addition to catalyzing the hydrolysis of CEs, hCEH also hydrolyzes TGs, and the murine homolog of hCEH, Ces3, has been characterized as Tgh and thought to play a role in VLDL secretion. To evaluate the effects of transgenic expression of hCEH, TG synthesis/secretion from precision cut liver slices was examined by monitoring incorporation of [3H]oleate in cellular as well as secreted TGs. There was no significant difference in [3H]oleate incorporation in cellular (tissue) as well as secreted (medium) TGs (Fig. 2A). Plasma TG levels were also measured and are shown in Fig. 2B. There was no significant difference in plasma TG levels with CEH over-expression (P = 0.76 for males, P = 0.79 for females). Consistently, the rates of in vivo secretion of VLDLs were also not significantly different between WT and LCEH2 mice (Fig. 2C). These data demonstrate that transgenic expression of hCEH does not affect hepatic TG synthesis or secretion. Cholesterol content of plasma lipoproteins also remained unchanged in LCEH2 mice (data not shown). To examine the role of hepatic CE hydrolysis in regulating elimination of HDL-derived CEs into bile, flux of [3H]cholesterol from HDL-CEs into bile was monitored in vivo. As shown in Fig. 3, transgenic expression of hCEH significantly increased the elimination of [3H]cholesterol from HDL-CEs to biliary bile acids (2.31 ± 1.05 vs. 1.14 ± 0.45, P = 0.03). In contrast, there was no increase in the [3H]label associated with biliary FC. These data suggest that FC generated by hepatic CE hydrolysis of HDL-CEs is preferentially eliminated as bile acids and is consistent with the observed increase in bile acid secretion by adenovirus-mediated transient over-expression of hCEH in mice (13Zhao B. Song J. Ghosh S. Hepatic overexpression of cholesteryl ester hydrolase enhances cholesterol elimination and in vivo reverse cholesterol transport.J. Lipid Res. 2008; 49: 2212-2217Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). LCEH2 mice were crossed into the LDLR−/− background and fed Western diet for 16 weeks and following parameters were examined. Hepatic expression of members of carboxylesterase family, as well as other known hydrolases, was examined and no significant differences were noted (supplementary Fig. V). Consistent with the data obtained in the C57BL/6 background, there was no significant change in the expression of genes involved in hepatic cholesterol homeostasis (HMG-CoAR, ACAT-2, Cyp7A1, and Cyp27A1) except that no significant increase in SR-BI expression was noted (supplementary Fig. VI). There was also no significant change in the expression of genes involved in cholesterol, phospholipid, and bile acid transport to the bile canaliculus, namely ABCCG5/G8, MDR2, and BSEP, respectively (supplementary Fig. VII). Fasting plasma TC and cholesterol associated with different lipoprotein fractions were measured and are shown in Fig. 4; representative fast-protein liquid chromatography profiles are shown in supplementary Fig. VIII. An overall significant effect of genotype (P = 0.008) and gender (P = 0.002) on TPC was detected by ANOVA (see Table 1). In addition, gender/genotype interaction was al