FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2043805010> ?p ?o ?g. }

• W2043805010 endingPage "326" @default.
• W2043805010 startingPage "312" @default.
• W2043805010 abstract "Liver X receptors (LXRs) are important regulators of cholesterol and lipid metabolism. LXR agonists have been shown to limit the cellular cholesterol content by inducing reverse cholesterol transport, increasing bile acid production, and inhibiting intestinal cholesterol absorption. Most of them, however, also increase lipogenesis via sterol regulatory element-binding protein-1c (SREBP1c) and carbohydrate response element-binding protein activation resulting in hypertriglyceridemia and liver steatosis. We report on the antiatherogenic properties of the steroidal liver X receptor agonist N,N-dimethyl-3β-hydroxy-cholenamide (DMHCA) in apolipoprotein E (apoE)-deficient mice. Long-term administration of DMHCA (11 weeks) significantly reduced lesion formation in male and female apoE-null mice. Notably, DMHCA neither increased hepatic triglyceride (TG) levels in male nor female apoE-deficient mice. ATP binding cassette transporter A1 and G1 and cholesterol 7α-hydroxylase mRNA abundances were increased, whereas SREBP1c mRNA expression was unchanged in liver, and even decreased in macrophages and intestine. Short-term treatment revealed even higher changes on mRNA regulation. Our data provide evidence that DMHCA is a strong candidate as therapeutic agent for the treatment or prevention of atherosclerosis, circumventing the negative side effects of other LXR agonists. Liver X receptors (LXRs) are important regulators of cholesterol and lipid metabolism. LXR agonists have been shown to limit the cellular cholesterol content by inducing reverse cholesterol transport, increasing bile acid production, and inhibiting intestinal cholesterol absorption. Most of them, however, also increase lipogenesis via sterol regulatory element-binding protein-1c (SREBP1c) and carbohydrate response element-binding protein activation resulting in hypertriglyceridemia and liver steatosis. We report on the antiatherogenic properties of the steroidal liver X receptor agonist N,N-dimethyl-3β-hydroxy-cholenamide (DMHCA) in apolipoprotein E (apoE)-deficient mice. Long-term administration of DMHCA (11 weeks) significantly reduced lesion formation in male and female apoE-null mice. Notably, DMHCA neither increased hepatic triglyceride (TG) levels in male nor female apoE-deficient mice. ATP binding cassette transporter A1 and G1 and cholesterol 7α-hydroxylase mRNA abundances were increased, whereas SREBP1c mRNA expression was unchanged in liver, and even decreased in macrophages and intestine. Short-term treatment revealed even higher changes on mRNA regulation. Our data provide evidence that DMHCA is a strong candidate as therapeutic agent for the treatment or prevention of atherosclerosis, circumventing the negative side effects of other LXR agonists. Nuclear liver X receptors (LXRs) are involved in the control of cholesterol and lipid metabolism. LXRα (NR1H3) and LXRβ (NR1H2) are sterol sensors that bind oxysterols to act as a transcriptional switch for the coordinated regulation of genes involved in cellular cholesterol homeostasis, cholesterol transport, catabolism, and absorption (1Janowski B.A Willy P.J. Devi T.R. Falck J.R. Mangelsdorf D.J. An oxysterol signalling pathway mediated by the nuclear receptor LXR alpha.Nature. 1996; 383: 728-731Crossref PubMed Scopus (1477) Google Scholar). In peripheral cells such as macrophages, LXRs are likely to coordinate a physiological response to cholesterol loading by regulating the transcription of several genes involved in cholesterol efflux and catabolism, including ATP-binding cassette (ABC)A1 and G1 (2Costet P. Luo Y. Wang N. Tall A.R. Sterol-dependent transactivation of the ABC1 promoter by the liver X receptor/retinoid X receptor.J. Biol. Chem. 2000; 275: 28240-28245Abstract Full Text Full Text PDF PubMed Scopus (853) Google Scholar, 3Repa J.J Turley S.D. Lobaccaro J.A. Medina J. Li L. Lustig K. Shan B. Heyman R.A. Dietschy J.M. Mangelsdorf D.J. Regulation of absorption and ABC1-mediated efflux of cholesterol by RXR heterodimers.Science. 2000; 289: 1524-1529Crossref PubMed Scopus (1151) Google Scholar, 4Schwartz K. Lawn R.M. Wade D.P. ABC1 gene expression and apoA-I-mediated cholesterol efflux are regulated by LXR.Biochem. Biophys. Res. Commun. 2000; 274: 794-802Crossref PubMed Scopus (376) Google Scholar, 5Venkateswaran A. Repa J.J. Lobaccaro J.M. Bronson A. Mangelsdorf D.J. Edwards P.A. Human white/murine ABC8 mRNA levels are highly induced in lipid-loaded macrophages. A transcriptional role for specific oxysterols.J. Biol. Chem. 2000; 275: 14700-14707Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 6Venkateswaran A. Laffitte B.A. Joseph S.B. Mak P.A. Wilpitz D.C. Edwards P.A. Tontonoz P. Control of cellular cholesterol efflux by the nuclear oxysterol receptor LXR alpha.Proc. Natl. Acad. Sci. USA. 2000; 97: 12097-12102Crossref PubMed Scopus (848) Google Scholar). In the intestine, LXR ligands were shown to reduce dietary cholesterol absorption by ABCA1, ABCG1, and ABCG5/G8 activation (3Repa J.J Turley S.D. Lobaccaro J.A. Medina J. Li L. Lustig K. Shan B. Heyman R.A. Dietschy J.M. Mangelsdorf D.J. Regulation of absorption and ABC1-mediated efflux of cholesterol by RXR heterodimers.Science. 2000; 289: 1524-1529Crossref PubMed Scopus (1151) Google Scholar, 7Brunham L.R Kruit J.K. Pape T.D. Parks J.S. Kuipers F. Hayden M.R. Tissue-specific induction of intestinal ABCA1 expression with a liver X receptor agonist raises plasma HDL cholesterol levels.Circ. Res. 2006; 99: 672-674Crossref PubMed Scopus (99) Google Scholar, 8Repa J.J Berge K.E. Pomajzl C. Richardson J.A. Hobbs H. Mangelsdorf D.J. Regulation of ATP-binding cassette sterol transporters ABCG5 and ABCG8 by the liver X receptors alpha and beta.J. Biol. Chem. 2002; 277: 18793-18800Abstract Full Text Full Text PDF PubMed Scopus (689) Google Scholar). In the liver, LXRs apparently regulate cholesterol, fatty acid, and triglyceride (TG) metabolism. This latter effect is partly mediated by sterol regulatory element-binding protein-1c (SREBP1c) activation (9Repa J.J Liang G. Ou J. Bashmakov Y. Lobaccaro J.M. Shimomura I. Shan B. Brown M.S. Goldstein J.L. Mangelsdorf D.J. Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRalpha and LXRbeta.Genes Dev. 2000; 14: 2819-2830Crossref PubMed Scopus (1423) Google Scholar, 10Yoshikawa T. Shimano H. Amemiya-Kudo M. Yahagi N. Hasty A.H. Matsuzaka T. Okazaki H. Tamura Y. Iizuka Y. Ohashi K. et al.Identification of liver X receptor-retinoid X receptor as an activator of the sterol regulatory element-binding protein 1c gene promoter.Mol. Cell. Biol. 2001; 21: 2991-3000Crossref PubMed Scopus (435) Google Scholar) and the upregulation of its downstream target genes [e.g., lipoprotein lipase (11Zhang Y. Repa J.J. Gauthier K. Mangelsdorf D.J. Regulation of lipoprotein lipase by the oxysterol receptors, LXRalpha and LXRbeta.J. Biol. Chem. 2001; 276: 43018-43024Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar), fatty acid synthase (FAS) (12Joseph S.B Laffitte B.A. Patel P.H. Watson M.A. Matsukuma K.E. Walczak R. Collins J.L. Osborne T.F. Tontonoz P. Direct and indirect mechanisms for regulation of fatty acid synthase gene expression by liver X receptors.J. Biol. Chem. 2002; 277: 11019-11025Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar), and stearoyl-CoA desaturase 1 (13Chu K. Miyazaki M. Man W.C. Ntambi J.M. Stearoyl-coenzyme A desaturase 1 deficiency protects against hypertriglyceridemia and increases plasma high-density lipoprotein cholesterol induced by liver X receptor activation.Mol. Cell. Biol. 2006; 26: 6786-6798Crossref PubMed Scopus (182) Google Scholar)]. Additionally, carbohydrate response element-binding protein (ChREBP) is activated by LXR (14Cha J.Y Repa J.J. The liver X receptor (LXR) and hepatic lipogenesis. The carbohydrate-response element-binding protein is a target gene of LXR.J. Biol. Chem. 2007; 282: 743-751Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar) and enhances hepatic fatty acid synthesis. In rodents but not in humans, LXRs enable bile acid synthesis by activation of cholesterol 7α-hydroxylase (CYP7A1) (15Lehmann J.M Kliewer S.A. Moore L.B. Smith-Oliver T.A. Oliver B.B. Su J.L. Sundseth S.S. Winegar D.A. Blanchard D.E. Spencer T.A. et al.Activation of the nuclear receptor LXR by oxysterols defines a new hormone response pathway.J. Biol. Chem. 1997; 272: 3137-3140Abstract Full Text Full Text PDF PubMed Scopus (1048) Google Scholar). Recent publications in atherosclerotic mouse models have shown that LXR activation by GW3965 and T0901317, two synthetic nonsteroidal LXR ligands, results in decreased atherosclerosis (16Joseph S.B McKilligin E. Pei L. Watson M.A. Collins A.R. Laffitte B.A. Chen M. Noh G. Goodman J. Hagger G.N. et al.Synthetic LXR ligand inhibits the development of atherosclerosis in mice.Proc. Natl. Acad. Sci. USA. 2002; 99: 7604-7609Crossref PubMed Scopus (787) Google Scholar, 17Terasaka N. Hiroshima A. Koieyama T. Ubukata N. Morikawa Y. Nakai D. Inaba T. T-0901317, a synthetic liver X receptor ligand, inhibits development of atherosclerosis in LDL receptor-deficient mice.FEBS Lett. 2003; 536: 6-11Crossref PubMed Scopus (293) Google Scholar). Unfortunately, the concomitant induction of lipogenic genes leads to hypertriglyceridemia and liver steatosis, which is an undesirable effect of most LXR agonists. N,N-dimethyl-3β-hydroxy-cholenamide (DMHCA) has been identified as a potent synthetic steroidal LXR activator in vitro and in vivo (18Quinet E.M Savio D.A. Halpern A.R. Chen L. Miller C.P. Nambi P. Gene-selective modulation by a synthetic oxysterol ligand of the liver X receptor.J. Lipid Res. 2004; 45: 1929-1942Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). It induces ABCA1 expression in macrophages and liver, but has only negligible effects on hepatic SREBP1c activation (approximately one-tenth of that observed for either T0901317 or GW3965) (18Quinet E.M Savio D.A. Halpern A.R. Chen L. Miller C.P. Nambi P. Gene-selective modulation by a synthetic oxysterol ligand of the liver X receptor.J. Lipid Res. 2004; 45: 1929-1942Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). These properties give DMHCA a novel and selective in vivo profile. In the present study, we have evaluated the effects of different doses and feeding periods of DMHCA in normal chow and Western type diet (WTD) on lipid parameters and gene regulation in wild-type mice. Furthermore, we determined whether DMHCA administration to apolipoprotein E (apoE)-deficient mice had an impact on atherosclerotic plaque formation. We found that DMHCA reduced atherosclerosis in male and female apoE-null mice without the development of hepatic steatosis and hypertriglyceridemia. These observations suggest that DMHCA may represent a promising therapeutic agent for intervention in atherosclerosis. Animal experiments were performed in accordance with the standards established by the Austrian Federal Ministry of Science and Research, Division of Genetic Engineering and Animal Experiments (Vienna, Austria). C57Bl/6 (Himberg, Austria) and apoE-deficient mice on a C57Bl/6 background (Charles River WIGA GesmbH, Sulzfeld, Germany) were maintained in a clean environment on a regular light-dark cycle (14 h light, 10 h dark). Before the initiation of the corresponding diets mice were kept on a standard laboratory chow diet. Male C57Bl/6 mice were fed chow diet or Western type diet (WTD; TD88137 mod. containing 21% fat and 0.2% cholesterol, ssniff, Soest, Germany and Harlan Teklad, Madison, WI) ± DMHCA or T0901317 (80 mg/kg body weight/day) (Cayman Chemicals, Ann Arbor, MI) for 4 or 15 days. To study atherosclerotic lesion formation, male and female apoE-deficient mice were fed ad libitum the WTD ± DMHCA (8 mg/kg body weight/day) for 11 weeks. To elucidate gene regulation by DMHCA in apoE-null mice in short time experiments, male mice were fed chow diet ± DMHCA (80 mg/kg body weight/day) for 4 days or WTD ± DMHCA (8 mg/kg body weight/day) for 15 days. Diets were supplemented with the respective LXR ligand at a level sufficient to provide the appropriate mg/kg food dose on consumption of a 5 g diet by a 25 g mouse per day. Body weight and food intake were monitored regularly. DMHCA was synthesized as described (19Louw D.F Strating G. Backer H.J. Delta-5-Steroids and provitamins D with branched side chains. III. Preparation and reduction of some delta-5-steroid omega-amines..Recueil des Travaux Chimiques des Pays-Bas et de la Belgique. 1954; 73: 667-676Crossref Scopus (5) Google Scholar) and/or was obtained from Dr. E. M. Quinet (18Quinet E.M Savio D.A. Halpern A.R. Chen L. Miller C.P. Nambi P. Gene-selective modulation by a synthetic oxysterol ligand of the liver X receptor.J. Lipid Res. 2004; 45: 1929-1942Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Purity was checked by thin layer chromatography and 1H-NMR. For analyses of atherosclerotic lesions at the aortic root, the upper two-thirds of the heart were fixed in 4% formaldehyde, embedded in tissue medium (Tissue-Tek O.C.T, Sanova Pharma GesmbH Diagnostik, Vienna, Austria), and frozen at −20°C. After approximately 600 μm, 8 μm cryosections of the aortic root were cut and lipid-rich regions were stained with Oil Red O (Sigma, Vienna, Austria) and counterstained with hemotoxylin (Richard-Allen Scientific, Kalamazoo, MI). Macrophages were detected with monoclonal rat anti-mouse antibody MOMA-2 (Acris, Hiddenhausen, Germany). Tissue specimens were fixed with acetone for 10 min at room temperature (RT). The slides were ultraviolet blocked and the sections were incubated with MOMA-2 antibody (1:600) for 30 min at RT. Subsequently, the sections were incubated with a biotinylated polyclonal rabbit anti-rat IgG (1:75, Dako Österreich GmbH, Vienna, Austria) for another 30 min at RT. The substrate chromogen AEC (Dako) was applied for 10 min at RT. The sections were counterstained with hemotoxylin. Images were taken with ScanScope T3 whole slide scanner (Aperio Technologies, Bristol, UK). Mean lesion area at the tricuspid valves was analyzed using Adobe Photoshop. For en face analysis, apoE-deficient mice were euthanized using 300 μl of Nembutal (diluted 1:5) and the heart was perfused for 10 min with PBS and for 15 min with 4% formaldehyde. Then the aorta was dissected, opened longitudinally from the heart to the iliac arteries, and stained with Oil Red O. Images were analyzed with Adobe Photoshop and a software algorithm which had been programmed with Interactive Data Language (ITT Visual Information Solutions, Boulder, CO). Briefly, the amount of plaques was calculated by the ratio Aplaque/Atotal after segmentation steps of the images with background and needle separation. The extent of lesion area is expressed as the percentage of the total area of aorta section covered by the lesion. Statistical analysis of the plaque area was performed using the statistic program SPSS 14.0, applying a Student's t-test. Nuclear extraction (20Wang X. Sato R. Brown M.S. Hua X. Goldstein J.L. SREBP-1, a membrane-bound transcription factor released by sterol-regulated proteolysis.Cell. 1994; 77: 53-62Abstract Full Text PDF PubMed Scopus (859) Google Scholar) and protein quantitation were performed as described (21Lowry O.H Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Aliquots (100 μg of protein) were separated by SDS-PAGE and blotted onto nitrocellulose membranes. For immunoblot analysis, SREBP1 antibody (Santa Cruz Biotechnology, Heidelberg, Germany) (1:1,000) was visualized with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:2,000, Dako Österreich GmbH, Vienna, Austria) using the Enhanced Chemiluminescence (ECL) Western Blotting Detection System Kit (Amersham Biosciences, GE Healthcare Europe GmbH, Vienna, Austria). Gels were calibrated with the Sigma marker wide range (Sigma, Vienna, Austria). Membranes were exposed to ECL Hyperfilm (Amersham Biosciences). Blood was collected by retro orbital bleeding and EDTA-plasma was prepared within 20 min. Plasma TG (DiaSys, Holzheim, Germany), total cholesterol (TC) (Greiner Diagnostics AG, Langenthal, Switzerland), HDL cholesterol (Technoclone GmbH, Vienna, Austria), alanine aminotransferase (ALT) (Roche Diagnostics, Mannheim, Germany) and aspartate aminotransferase (AST) (Thermo Electron Corporation, Louisville, CO) concentrations were measured enzymatically. To determine hepatic lipid contents, total lipids were extracted from livers and lipid parameters were determined using above mentioned kits. For analysis of hepatic TG-associated fatty acids lipids from livers were extracted and separated by TLC. The TG band was scraped, methylated, and analyzed by gas-liquid chromatography using heptadecanoic acid as internal standard (22Miyazaki M. Kim H.J. Man W.C. Ntambi J.M. Oleoyl-CoA is the major de novo product of stearoyl-CoA desaturase 1 gene isoform and substrate for the biosynthesis of the Harderian gland 1-alkyl-2,3-diacylglycerol..J. Biol. Chem. 2001; 276: 39455-39461Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Plasma samples of seven overnight fasted female apoE-deficient mice fed WTD plus DMHCA for 11 weeks were pooled and compared with those of seven apoE-deficient mice fed WTD (control). Lipoproteins were isolated by fast protein liquid chromatography (FPLC) on a Pharmacia FPLC system (Pfizer Pharma, Karlsruhe, Germany) equipped with a Superose 6 column (Amersham Biosciences, Piscataway, NJ). Two hundred μl pooled plasma samples were diluted, subjected to FPLC analysis and lipoproteins were eluted with 10 mM Tris-HCl, 1 mM EDTA, 0.9% NaCl, and 0.02% NaN3 (pH 7.4). Fractions of 0.5 ml each were collected and TG and TC concentrations were assayed enzymatically using above mentioned kit. To enhance sensitivity, reaction buffers were supplemented by the addition of sodium 3,5-dichloro-2-hydroxy-benzenesulfonate (Sigma-Aldrich, Vienna, Austria). Thioglycollate-elicited peritoneal macrophages (MPM) were isolated with 10 ml PBS from mice 3 days after peritoneal injection of 3 ml 3% thioglycollate medium. MPM were centrifuged, washed with PBS, and cultured in 75 cm2 flasks in DMEM (Gibco, Invitrogen, Lofer, Austria) supplemented with 10% FCS, 1% L-glutamine, and 1% streptomycin/penicillin under standard cell culture conditions (37°C, 5% CO2) for 2 h. Thereafter, nonadherent cells were removed and MPM were cultured in the same medium overnight. Macrophage differentiation to foam cells (from C57Bl/6 mice) was performed by incubation of MPM with 50 μg/ml aggregated LDL for 24 h in the absence or presence of 2.5 μM DMHCA. Total RNA from mouse tissues was isolated using the Trizol procedure according to the manufacturer's protocol (Invitrogen, Lofer, Austria). Total RNA from cells was isolated using RNeasy Mini Kit (Qiagen, Vienna, Austria). Quantitative gene expression analysis was performed on a LightCycler 480 (Roche Diagnostics, Mannheim, Germany) using the Quantifast™ SYBR®GREEN PCR Kit (Qiagen). For RNA quantification, 1–2 μg of total RNA were reverse transcribed according to the manufacturer's instructions using random hexamer primers (Finnzymes, Espoo, Finland). In general, 6 ng template cDNA was used for each real time PCR. PCR primers used for real-time PCR (see supplementary information) were designed with primer designer version 2.0. Melting curve analysis was performed to ensure that a single PCR product was amplified and no primer dimers were generated. PCR efficiency of each transcript was determined using four dilutions (1:5, 1:25, 1:125, 1:625) in triplicate of a pool of all available cDNAs of one experiment. To confirm accuracy and reproducibility a minimum of three samples per condition were measured in triplicate. Crossing points were determined by Second Derivate Maximum Method and PCR efficiencies were calculated from the slope, according to the established equation E = 10[-1/slope]. Data calculations and determination of statistical parameters were performed with the public domain program relative expression software tool REST (http://www.gene-quantification.com/download.html) using a pair-wise fixed reallocation randomization test (23Pfaffl M.W Horgan G.W. Dempfle L. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR.Nucleic Acids Res. 2002; 30: e36Crossref PubMed Google Scholar). Data are displayed as expression ratios normalized to a reference gene. Initially, amplifications of murine cyclophilin A were performed as internal controls for variations in mRNA amounts. In the liver, cyclophilin A expression varied between mouse genotypes and therefore hypoxanthine guanine phosphoribosyl transferase (HPRT) was used as reference gene. In macrophages, aorta, and ileum data are displayed as expression ratios normalized to cyclophilin A as reference gene. Statistical analyses in experiments except real-time PCR analyses (see above) were performed using the Student's t-test. Data are expressed as mean ± SD. * P < 0.05; ** P ≤ 0.01; *** P ≤ 0.001. MPM isolated from C57Bl/6 mice and foam cells were incubated in the absence or presence of 2.5 μM DMHCA for 24 h. As expected, DMHCA was found to stimulate ABCA1 mRNA expression in MPM and in aggregated LDL-laden foam cells in vitro by 7.0- and 4.8-fold, respectively, compared with untreated macrophages and foam cells (Fig. 1). To study possible effects of a short-term treatment, C57Bl/6 mice were fed chow diet containing a high dose of either DMHCA or T0901317 (80 mg/kg body weight/day) for 4 days. Although all mice ate comparable amounts of food and showed similar body weights, liver weights were significantly increased in T0901317-fed animals (1.54 ± 0.24 g) when compared with controls (1.12 ± 0.12 g) (Fig. 2A). Macroscopically, administration of T0901317 resulted in a yellow-brown change in color of the liver and moderate to severe steatosis (Fig. 2A). This effect was also confirmed by measuring the TG concentration, which was significantly increased by 2.4-fold when compared with controls (Fig. 2, inset). In addition, plasma alanine aminotransferase (ALT) concentration was increased (241 ± 95 U/l) indicating liver damage following treatment with T0901317 (Fig. 2, inset). Furthermore, T0901317 treatment significantly enhanced SREBP1c mRNA levels by 4.4-fold (Fig. 2B). There were no differences in the content of the precursor SREBP1 protein between the different diets, however, T0901317 markedly increased mature, transcriptionally active hepatic nuclear SREBP1 (nSREBP1) protein by 2.9-fold (Fig. 2C). In contrast, liver weight remained unchanged (1.15 ± 0.04 g) and the liver color appeared normal in DMHCA-fed mice (Fig. 2A). Oil Red O staining of liver specimens from DMHCA-treated animals revealed minimal steatosis (Fig. 2A) with slightly increased hepatic TG levels (24.5 ± 4.8 mg/g) when compared with controls (19.7 ± 1.6 mg/g) (Fig. 2, inset). In accordance, DMHCA treatment resulted in only 1.6-fold increased SREBP1c mRNA (Fig. 2B) and there were no changes in nSREBP1 protein levels (Fig. 2C). Plasma ALT levels were not significantly altered (Fig. 2, inset). These data clearly demonstrate that T0901317 had a profound effect on hepatic fat content with a drastic increase in plasma ALT levels after 4 days of feeding, while DMHCA only mildly increased hepatic TG concentrations with no detectable liver damage. To check whether a treatment longer than 4 days or WTD affects lipid parameters, male C57Bl/6 mice were fed chow or WTD ± 80 mg DMHCA/kg body weight/day for 15 days. Plasma TG concentrations were not significantly altered by DMHCA on both diets suggesting that a high dose of DMHCA fed for more than 2 weeks does not induce hypertriglyceridemia in wild-type animals (Table 1). Interestingly, plasma TC levels were significantly reduced by 32% in mice fed chow diet plus DMHCA but remained unchanged in mice fed WTD plus DMHCA when compared with controls. Hepatic TC concentrations were markedly decreased on both diets by DMHCA (54% and 56%, respectively). In contrast, hepatic TG levels were not changed by DMHCA in WTD, but were significantly increased by 2.4-fold by DMHCA in chow diet. Plasma AST levels were slightly but not significantly increased on chow diet plus DMHCA and unaltered by DMHCA in WTD-fed animals (Table 1). Oil Red O staining of liver sections from DMHCA-treated mice revealed macroscopically no differences in lipid accumulation upon the absence or presence of DMHCA in both diets, while T0901317 treatment resulted in severe steatosis with enlarged lipid droplets on both diets (see supplementary Figure I). Because we found slightly increased TG levels in DMHCA-treated livers on chow diet, we checked the hepatic fatty acid composition in the TG fraction, which was changed upon high DMHCA concentrations in the chow diet. Oleate (C18:1), palmitate (C16:0), linoleate (C18:2), and stearate (C16:1) were significantly increased (Fig. 3A). No alterations were observed in mice fed WTD plus DMHCA compared with WTD alone (Fig. 3B).TABLE 1Plasma and hepatic triglyceride and total cholesterol concentrations of mice fed N,N-dimethyl-3β-hydroxy-cholenamide (DMHCA) for 15 daysPlasmaLiverTG (mg/dl)TC (mg/dl)AST (IU/l)TG (mg/g tissue)TC (mg/g tissue)Chow62.3 ± 10.3127 ± 16.430.1 ± 13.014.0 ± 6.42.16 ± 0.40Chow + DMHCA52.6 ± 8.286.8 ± 15.5bP ≤ 0.001.47.7 ± 13.233.8 ± 14.9aP < 0.05.1.00 ± 0.45bP ≤ 0.001.WTD45.6 ± 8.1208 ± 65.926.7 ± 8.732.1 ± 12.84.28 ± 0.55WTD + DMHCA54.9 ± 10.8217 ± 50.621.4 ± 7.335.1 ± 11.11.88 ± 0.44bP ≤ 0.001.AST, aspartate aminotransferase; DMHCA, N,N-dimethyl-3β-hydroxy-cholenamide. Plasma and hepatic triglyceride (TG) and total cholesterol (TC) concentrations of male C57Bl/6 mice fed chow or Western type diet (WTD) ± N,N-dimethyl-3β-hydroxy-cholenamide (DMHCA) (80 mg/kg body weight/day) for 15 days. Data are expressed as mean ± SD values of 6-8 mice aged 8–12 weeks.a P < 0.05.b P ≤ 0.001. Open table in a new tab AST, aspartate aminotransferase; DMHCA, N,N-dimethyl-3β-hydroxy-cholenamide. Plasma and hepatic triglyceride (TG) and total cholesterol (TC) concentrations of male C57Bl/6 mice fed chow or Western type diet (WTD) ± N,N-dimethyl-3β-hydroxy-cholenamide (DMHCA) (80 mg/kg body weight/day) for 15 days. Data are expressed as mean ± SD values of 6-8 mice aged 8–12 weeks. Target gene expression levels in livers of C57Bl/6 mice fed chow or WTD ± T0901317 or DMHCA (80 mg/kg body weight/day) for 15 days were analyzed by real time PCR. In chow diet, treatment with DMHCA led to a significant 2.5-fold increase of CYP7A1 mRNA expression (Fig. 4A). Additionally, the expression levels of ABCA1, ABCG1, ABCG5, and ABCG8 were significantly increased by 1.9-, 2.1-, 3.5- and 2.8-fold, respectively. Hepatic SREBP1c, fatty acid synthase (FAS) and ChREBP mRNA quantities were unaltered upon DMHCA treatment. T0901317 in chow diet resulted in more pronounced increase of CYP7A1, ABCA1, ABCG1, and ABCG5/G8 mRNA expression compared with controls, but also enhanced SREBP1c, FAS, and ChREBP mRNA by 4.7-, 3.4-, and 1.3-fold, respectively (Fig. 4A). Similar real-time PCR results were obtained when mRNA levels were determined from livers of mice fed WTD plus the LXR agonists. Both ligands significantly increased CYP7A1 and ABCG8 mRNA, only T0901317 resulted in an increase in ABCG1 and ABCG5 mRNA, while ABCA1 mRNA was unaltered by T0901317 and DMHCA treatment. While T0901317 significantly increased SREBP1c and FAS by 3.4- and 47.3-fold, respectively, DMHCA did not alter SREBP1c mRNA expression. While FAS mRNA was significantly increased by 2.7-fold, ChREBP was significantly decreased upon DMHCA treatment by 47%. We also analyzed ABCA1, ABCG1, ABCG5, and ABCG8 mRNA levels in ileum of T0901317 and DMHCA in chow diet and found these ABC transporters significantly increased by both ligands; again the upregulation was higher by T0901317 treatment (see supplementary Figure II). Because we observed beneficial effects of DMHCA in wild-type mice, we further investigated the effect of DMHCA on body weight and lipid parameters in apoE-deficient mice during a long-term treatment. For that purpose, animals were fed WTD with or without DMHCA (8 mg/kg body weight/day) for 11 weeks. Although the food intake was similar in the different groups, male and female mice fed WTD plus DMHCA showed slightly decreased body weight gain when compared with mice fed WTD without DMHCA, which reached significance in female mice after 8 weeks of treatment (Fig. 5A,B). Plasma TC and TG levels were determined biweekly in the fed state. While no significant differences were observed in plasma TC (Fig. 5C) and TG (Fig. 5E) concentrations in male mice, female mice showed a significant decrease in TC (Fig. 5D) and TG (Fig. 5F) levels. Similar changes in plasma lipid levels were observed when mice were fasted overnight before blood was taken after 11 weeks of feeding WTD plus DMHCA (Table 2). We found reduced plasma TG levels in male (140 ± 10 mg/dl) and female (163 ± 9 mg/dl) apoE-deficient mice compared with mice fed WTD alone (197 ± 26 and 220 ± 5 mg/dl, respectively), whereas TC concentrations were significantly decreased only in male (799 ± 65 mg/dl vs. 1131 ± 52 mg/dl) but not in female animals (587 ± 33 mg/dl vs. 638 ± 16 mg/dl, respectively). No significant changes were found in HDL cholesterol levels. Determination of plasma AST or ALT levels did not give reliable results due to lipemic samples. Yet we determined bilirubin concentrations and found that they were comparable upon all diets. Therefore, long-time treatment did not lead to liver damage in apoE-deficient mice. A" @default.
• W2043805010 created "2016-06-24" @default.
• W2043805010 creator A5003449456 @default.
• W2043805010 creator A5005767097 @default.
• W2043805010 creator A5009385227 @default.
• W2043805010 creator A5010830962 @default.
• W2043805010 creator A5023340444 @default.
• W2043805010 creator A5026066712 @default.
• W2043805010 creator A5031807071 @default.
• W2043805010 creator A5032236530 @default.
• W2043805010 creator A5032849794 @default.
• W2043805010 creator A5038675534 @default.
• W2043805010 creator A5039920851 @default.
• W2043805010 creator A5041918931 @default.
• W2043805010 creator A5046244856 @default.
• W2043805010 creator A5049644118 @default.
• W2043805010 creator A5055551947 @default.
• W2043805010 creator A5057840359 @default.
• W2043805010 creator A5059534716 @default.
• W2043805010 creator A5063982521 @default.
• W2043805010 creator A5075187158 @default.
• W2043805010 creator A5075328733 @default.
• W2043805010 creator A5081349755 @default.
• W2043805010 creator A5083292403 @default.
• W2043805010 date "2009-02-01" @default.
• W2043805010 modified "2023-10-17" @default.
• W2043805010 title "Synthetic LXR agonist attenuates plaque formation in apoE-/- mice without inducing liver steatosis and hypertriglyceridemia" @default.
• W2043805010 cites W1510167055 @default.
• W2043805010 cites W1570388980 @default.
• W2043805010 cites W1607230014 @default.
• W2043805010 cites W1775749144 @default.
• W2043805010 cites W1964532888 @default.
• W2043805010 cites W1967286688 @default.
• W2043805010 cites W1973243569 @default.
• W2043805010 cites W1996168740 @default.
• W2043805010 cites W2003840075 @default.
• W2043805010 cites W2009577529 @default.
• W2043805010 cites W2021632088 @default.
• W2043805010 cites W2023345325 @default.
• W2043805010 cites W2024851295 @default.
• W2043805010 cites W2026587556 @default.
• W2043805010 cites W2026670066 @default.
• W2043805010 cites W2037070298 @default.
• W2043805010 cites W2039928578 @default.
• W2043805010 cites W2044842369 @default.
• W2043805010 cites W2048397414 @default.
• W2043805010 cites W2050798797 @default.
• W2043805010 cites W2053365325 @default.
• W2043805010 cites W2085909854 @default.
• W2043805010 cites W2093296161 @default.
• W2043805010 cites W2094753712 @default.
• W2043805010 cites W2095987780 @default.
• W2043805010 cites W2099249565 @default.
• W2043805010 cites W2099378835 @default.
• W2043805010 cites W2101284986 @default.
• W2043805010 cites W2112460321 @default.
• W2043805010 cites W2113668616 @default.
• W2043805010 cites W2128660079 @default.
• W2043805010 cites W2135007868 @default.
• W2043805010 cites W2153673003 @default.
• W2043805010 cites W2157038552 @default.
• W2043805010 cites W2159131813 @default.
• W2043805010 cites W2159332620 @default.
• W2043805010 cites W2332525086 @default.
• W2043805010 cites W4210363776 @default.
• W2043805010 doi "https://doi.org/10.1194/jlr.m800376-jlr200" @default.
• W2043805010 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2636920" @default.
• W2043805010 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18812595" @default.
• W2043805010 hasPublicationYear "2009" @default.
• W2043805010 type Work @default.
• W2043805010 sameAs 2043805010 @default.
• W2043805010 citedByCount "123" @default.
• W2043805010 countsByYear W20438050102012 @default.
• W2043805010 countsByYear W20438050102013 @default.
• W2043805010 countsByYear W20438050102014 @default.
• W2043805010 countsByYear W20438050102015 @default.
• W2043805010 countsByYear W20438050102016 @default.
• W2043805010 countsByYear W20438050102017 @default.
• W2043805010 countsByYear W20438050102018 @default.
• W2043805010 countsByYear W20438050102019 @default.
• W2043805010 countsByYear W20438050102020 @default.
• W2043805010 countsByYear W20438050102021 @default.
• W2043805010 countsByYear W20438050102022 @default.
• W2043805010 countsByYear W20438050102023 @default.
• W2043805010 crossrefType "journal-article" @default.
• W2043805010 hasAuthorship W2043805010A5003449456 @default.
• W2043805010 hasAuthorship W2043805010A5005767097 @default.
• W2043805010 hasAuthorship W2043805010A5009385227 @default.
• W2043805010 hasAuthorship W2043805010A5010830962 @default.
• W2043805010 hasAuthorship W2043805010A5023340444 @default.
• W2043805010 hasAuthorship W2043805010A5026066712 @default.
• W2043805010 hasAuthorship W2043805010A5031807071 @default.
• W2043805010 hasAuthorship W2043805010A5032236530 @default.
• W2043805010 hasAuthorship W2043805010A5032849794 @default.
• W2043805010 hasAuthorship W2043805010A5038675534 @default.
• W2043805010 hasAuthorship W2043805010A5039920851 @default.
• W2043805010 hasAuthorship W2043805010A5041918931 @default.
• W2043805010 hasAuthorship W2043805010A5046244856 @default.

• next