Lack of stimulation of cholesteryl ester transfer protein by cholesterol in the presence of a high-fat diet
2005; Elsevier BV; Volume: 46; Issue: 11 Linguagem: Inglês
10.1194/jlr.m500051-jlr200
ISSN1539-7262
AutoresSukhinder Kaur Cheema, Alka Agarwal-Mawal, Cathy Murray, Stephanie C. Tucker,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoCholesteryl ester transfer protein (CETP) is a key protein involved in the reverse cholesterol transport pathway. The regulation of CETP by dietary fats is not clearly understood. Transgenic mice expressing human CETP under the control of its natural flanking region were fed low- or high-fat diets enriched in monounsaturated fatty acids (MUFAs) or saturated fatty acids in the presence or absence of cholesterol. Addition of cholesterol to the low-fat MUFA diet increased CETP activity and mRNA expression, whereas addition of cholesterol to the high-fat MUFA diet led to a decrease in CETP activity and mRNA expression. In SW 872 cells, oleic acid and cholesterol stimulated CETP gene expression when given alone. However, addition of fatty acids along with cholesterol interfered with the stimulatory effect of cholesterol on CETP gene regulation. Cholesterol-mediated stimulation of CETP involves the transcription factor liver X receptor α (LXRα). High-fat MUFA diets inhibited the expression of LXRα, and addition of cholesterol to the high-fat MUFA diet did not rescue LXRα expression.Therefore, we present evidence for the first time that inhibition of LXRα expression by a high-fat MUFA diet leads to inhibition of CETP stimulation by cholesterol. Cholesteryl ester transfer protein (CETP) is a key protein involved in the reverse cholesterol transport pathway. The regulation of CETP by dietary fats is not clearly understood. Transgenic mice expressing human CETP under the control of its natural flanking region were fed low- or high-fat diets enriched in monounsaturated fatty acids (MUFAs) or saturated fatty acids in the presence or absence of cholesterol. Addition of cholesterol to the low-fat MUFA diet increased CETP activity and mRNA expression, whereas addition of cholesterol to the high-fat MUFA diet led to a decrease in CETP activity and mRNA expression. In SW 872 cells, oleic acid and cholesterol stimulated CETP gene expression when given alone. However, addition of fatty acids along with cholesterol interfered with the stimulatory effect of cholesterol on CETP gene regulation. Cholesterol-mediated stimulation of CETP involves the transcription factor liver X receptor α (LXRα). High-fat MUFA diets inhibited the expression of LXRα, and addition of cholesterol to the high-fat MUFA diet did not rescue LXRα expression. Therefore, we present evidence for the first time that inhibition of LXRα expression by a high-fat MUFA diet leads to inhibition of CETP stimulation by cholesterol. Cholesteryl ester transfer protein (CETP) is considered a key component in regulating cholesterol homeostasis as it transfers cholesteryl esters from HDL to apolipoprotein B-containing lipoproteins (1Bruce C. Chouinard Jr., R.A. Tall A.R. Plasma lipid transfer proteins, high density lipoproteins, and reverse cholesterol transport.Annu. Rev. Nutr. 1998; 18: 297-330Crossref PubMed Scopus (229) Google Scholar, 2Fielding C.J. Fielding P.E. Molecular physiology of reverse cholesterol transport.J. Lipid Res. 1995; 36: 211-228Abstract Full Text PDF PubMed Google Scholar, 3Foger B. Ritsch A. Doblinger A. Wessels H. Patsch J.R. Relationship of plasma cholesteryl ester transfer protein to HDL cholesterol. Studies in normotriglyceridemia and moderate hypertriglyceridemia.Arterioscler. Thromb. Vasc. Biol. 1996; 16: 1430-1436Crossref PubMed Scopus (67) Google Scholar, 4Martin L.J. Connelly P.W. Nancoo D. Wood N. Zhang Z.J. Maguire G. Quinet E. Tall A.R. Marcel Y.L. McPherson R. Cholesteryl ester transfer protein and high density lipoprotein responses to cholesterol feeding in men: relationship to apolipoprotein E genotype.J. Lipid Res. 1993; 34: 437-446Abstract Full Text PDF PubMed Google Scholar). Increased plasma CETP levels result in low plasma HDL and high plasma LDL or VLDL levels (5Takahashi H. Takahashi A. Maki M. Sasai H. Kamada M. Effect of CETP on the plasma lipoprotein profile in four strains of transgenic mouse.Biochem. Biophys. Res. Commun. 2001; 283: 118-123Crossref PubMed Scopus (13) Google Scholar, 6Hogue J.C. Lamarche B. Gaudet D. Lariviere M. Tremblay A.J. Bergeron J. Lemieux I. Despres J.P. Gagne C. Couture P. Relationship between cholesteryl ester transfer protein and LDL heterogeneity in familial hypercholesterolemia.J. Lipid Res. 2004; 45: 1077-1083Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). HDL plays a major role in reverse cholesterol transport, a process that involves the movement of cholesterol from peripheral cells to the liver for removal from the body. Studies in transgenic mice, and in humans with alterations in CETP levels, show that an absence of CETP is associated with the disruption of cholesterol efflux from cell membranes, of cholesterol esterification, and of cholesteryl ester transfer to apolipoprotein B-containing lipoproteins (1Bruce C. Chouinard Jr., R.A. Tall A.R. Plasma lipid transfer proteins, high density lipoproteins, and reverse cholesterol transport.Annu. Rev. Nutr. 1998; 18: 297-330Crossref PubMed Scopus (229) Google Scholar). These disruptions impair cholesterol movement to the liver; thus, a high CETP activity might be antiatherogenic as it removes excess cholesterol from the arterial wall. On the other hand, CETP produces VLDL particles that are cholesteryl ester-enriched and decreases the level of antiatherogenic HDL cholesterol; thus, CETP is considered proatherogenic (1Bruce C. Chouinard Jr., R.A. Tall A.R. Plasma lipid transfer proteins, high density lipoproteins, and reverse cholesterol transport.Annu. Rev. Nutr. 1998; 18: 297-330Crossref PubMed Scopus (229) Google Scholar, 2Fielding C.J. Fielding P.E. Molecular physiology of reverse cholesterol transport.J. Lipid Res. 1995; 36: 211-228Abstract Full Text PDF PubMed Google Scholar, 3Foger B. Ritsch A. Doblinger A. Wessels H. Patsch J.R. Relationship of plasma cholesteryl ester transfer protein to HDL cholesterol. Studies in normotriglyceridemia and moderate hypertriglyceridemia.Arterioscler. Thromb. Vasc. Biol. 1996; 16: 1430-1436Crossref PubMed Scopus (67) Google Scholar, 4Martin L.J. Connelly P.W. Nancoo D. Wood N. Zhang Z.J. Maguire G. Quinet E. Tall A.R. Marcel Y.L. McPherson R. Cholesteryl ester transfer protein and high density lipoprotein responses to cholesterol feeding in men: relationship to apolipoprotein E genotype.J. Lipid Res. 1993; 34: 437-446Abstract Full Text PDF PubMed Google Scholar). In humans, CETP deficiency is associated with high HDL levels and a low prevalence of coronary heart disease (7Brown M.L. Inazu A. Hesler C.B. Agellon L.B. Mann C. Whitlock M.E. Marcel Y.L. Milne R.W. Koizumi J. Mabuchi H. et al.Molecular basis of lipid transfer protein deficiency in a family with increased high-density lipoproteins.Nature. 1989; 342: 448-451Crossref PubMed Scopus (411) Google Scholar, 8Inazu A. 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Reduced high density lipoprotein cholesterol in human cholesteryl ester transfer protein transgenic mice.J. Biol. Chem. 1991; 266: 10796-10801Abstract Full Text PDF PubMed Google Scholar) or simian (10Marotti K.R. Castle C.K. Boyle T.P. Lin A.H. Murray R.W. Melchior G.W. Severe atherosclerosis in transgenic mice expressing simian cholesteryl ester transfer protein.Nature. 1993; 364: 73-75Crossref PubMed Scopus (415) Google Scholar) CETP exhibit a marked reduction in HDL cholesterol, also suggesting that high levels of CETP protein impart increased risk of coronary artery disease. Plasma CETP activity increases in response to high-fat, high-cholesterol diets in hamsters (11Jiang X.C. Moulin P. Quinet E. Goldberg I.J. Yacoub L.K. Agellon L.B. Compton D. Schnitzer-Polokoff R. Tall A.R. Mammalian adipose tissue and muscle are major sources of lipid transfer protein mRNA.J. Biol. Chem. 1991; 266: 4631-4639Abstract Full Text PDF PubMed Google Scholar), rabbits (12Son Y.S. Zilversmit D.B. Increased lipid transfer activities in hyperlipidemic rabbit plasma.Arteriosclerosis. 1986; 6: 345-351Crossref PubMed Google Scholar, 13McPherson R. Lau P. Kussie P. Barrett H. Tall A.R. Plasma kinetics of cholesteryl ester transfer protein in the rabbit. Effects of dietary cholesterol.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 203-210Crossref PubMed Scopus (22) Google Scholar), monkeys (14Quinet E. Tall A. Ramakrishnan R. Rudel L. Plasma lipid transfer protein as a determinant of the atherogenicity of monkey plasma lipoproteins.J. Clin. Invest. 1991; 87: 1559-1566Crossref PubMed Scopus (140) Google Scholar, 15Khosla P. Hajri T. Pronczuk A. Hayes K.C. Replacing dietary palmitic acid with elaidic acid (t-C18:1 delta9) depresses HDL and increases CETP activity in cebus monkeys.J. Nutr. 1997; 127: 531-536Crossref Google Scholar, 16Fusegawa Y. Kelley K.L. Sawyer J.K. Shah R.N. Rudel L.L. Influence of dietary fatty acid composition on the relationship between CETP activity and plasma lipoproteins in monkeys.J. Lipid Res. 2001; 42: 1849-1857Abstract Full Text Full Text PDF PubMed Google Scholar), and humans (4Martin L.J. Connelly P.W. Nancoo D. Wood N. Zhang Z.J. Maguire G. Quinet E. Tall A.R. Marcel Y.L. McPherson R. Cholesteryl ester transfer protein and high density lipoprotein responses to cholesterol feeding in men: relationship to apolipoprotein E genotype.J. Lipid Res. 1993; 34: 437-446Abstract Full Text PDF PubMed Google Scholar) and also in transgenic mice expressing human CETP (17Jiang X.C. Agellon L.B. Walsh A. Breslow J.L. Tall A. Dietary cholesterol increases transcription of the human cholesteryl ester transfer protein gene in transgenic mice. Dependence on natural flanking sequences.J. Clin. Invest. 1992; 90: 1290-1295Crossref PubMed Google Scholar, 18Chang C. Snook J.T. The cholesterolaemic effects of dietary fats in cholesteryl ester transfer protein transgenic mice.Br. J. Nutr. 2001; 85: 643-648Crossref PubMed Google Scholar). The increase in CETP activity in response to an atherogenic diet is associated with an increase in hepatic mRNA abundance (19Quinet E.M. Agellon L.B. Kroon P.A. Marcel Y.L. Lee Y.C. Tall A.R. Atherogenic diet increases cholesteryl ester transfer protein messenger RNA levels in rabbit liver.J. Clin. Invest. 1990; 85: 357-363Crossref PubMed Scopus (112) Google Scholar), which in CETP transgenic mice fully accounts for the increase in CETP activity (17Jiang X.C. Agellon L.B. Walsh A. Breslow J.L. Tall A. Dietary cholesterol increases transcription of the human cholesteryl ester transfer protein gene in transgenic mice. Dependence on natural flanking sequences.J. Clin. Invest. 1992; 90: 1290-1295Crossref PubMed Google Scholar). Although dietary cholesterol is likely to be the major factor contributing to an increase in plasma CETP activity, synthetic diets that lead to increased hypercholesterolemia, without the addition of cholesterol, also result in a similar increase in plasma CETP activity (20Kurushima H. Hayashi K. Shingu T. Kuga Y. Ohtani H. Okura Y. Tanaka K. Yasunobu Y. Nomura K. Kajiyama G. Opposite effects on cholesterol metabolism and their mechanisms induced by dietary oleic acid and palmitic acid in hamsters.Biochim. Biophys. Acta. 1995; 1258: 251-256Crossref PubMed Scopus (38) Google Scholar). There are very few reports available to show the effect of dietary fats on CETP regulation (16Fusegawa Y. Kelley K.L. Sawyer J.K. Shah R.N. Rudel L.L. Influence of dietary fatty acid composition on the relationship between CETP activity and plasma lipoproteins in monkeys.J. Lipid Res. 2001; 42: 1849-1857Abstract Full Text Full Text PDF PubMed Google Scholar, 18Chang C. Snook J.T. The cholesterolaemic effects of dietary fats in cholesteryl ester transfer protein transgenic mice.Br. J. Nutr. 2001; 85: 643-648Crossref PubMed Google Scholar, 20Kurushima H. Hayashi K. Shingu T. Kuga Y. Ohtani H. Okura Y. Tanaka K. Yasunobu Y. Nomura K. Kajiyama G. Opposite effects on cholesterol metabolism and their mechanisms induced by dietary oleic acid and palmitic acid in hamsters.Biochim. Biophys. Acta. 1995; 1258: 251-256Crossref PubMed Scopus (38) Google Scholar, 21Groener J.E. van Ramshorst E.M. Katan M.B. Mansink R.P. Tol A. van Diet induced alteration in the activity of plasma lipid transfer protein in normolipidemic human subjects.Atherosclerosis. 1991; 87: 221-226Abstract Full Text PDF PubMed Scopus (51) Google Scholar, 22Kurushima H. Hayashi K. Toyota Y. Kambe M. Kajiyama G. Comparison of hypocholesterolemic effects induced by dietary linoleic acid and oleic acid in hamsters.Atherosclerosis. 1995; 114: 213-221Abstract Full Text PDF PubMed Scopus (32) Google Scholar, 23Jansen S. Lopez-Miranda J. Castro P. Lopez-Segura F. Marin C. Ordovas J.M. Paz E. Jimenez-Pereperez J. Fuentes F. Perez-Jimenez F. Low-fat and high-monounsaturated fatty acid diets decrease plasma cholesterol ester transfer protein concentrations in young, healthy, normolipemic men.Am. J. Clin. Nutr. 2000; 72: 36-41Crossref PubMed Scopus (61) Google Scholar, 24Gupta S.V. Yamada N. Fungwe T.V. Khosla P. Replacing 40% of dietary animal fat with vegetable oil is associated with lower HDL cholesterol and higher cholesterol ester transfer protein in cynomolgus monkeys fed sufficient linoleic acid.J. Nutr. 2003; 133: 2600-2606Crossref PubMed Scopus (6) Google Scholar). Although saturated fatty acids (SFAs) have been consistently shown to increase CETP, the effects observed with unsaturated fatty acids are variable. Reports indicate that monounsaturated fatty acids (MUFAs) decrease (20Kurushima H. Hayashi K. Shingu T. Kuga Y. Ohtani H. Okura Y. Tanaka K. Yasunobu Y. Nomura K. Kajiyama G. Opposite effects on cholesterol metabolism and their mechanisms induced by dietary oleic acid and palmitic acid in hamsters.Biochim. Biophys. Acta. 1995; 1258: 251-256Crossref PubMed Scopus (38) Google Scholar, 21Groener J.E. van Ramshorst E.M. Katan M.B. Mansink R.P. Tol A. van Diet induced alteration in the activity of plasma lipid transfer protein in normolipidemic human subjects.Atherosclerosis. 1991; 87: 221-226Abstract Full Text PDF PubMed Scopus (51) Google Scholar, 23Jansen S. Lopez-Miranda J. Castro P. Lopez-Segura F. Marin C. Ordovas J.M. Paz E. Jimenez-Pereperez J. Fuentes F. Perez-Jimenez F. Low-fat and high-monounsaturated fatty acid diets decrease plasma cholesterol ester transfer protein concentrations in young, healthy, normolipemic men.Am. J. Clin. Nutr. 2000; 72: 36-41Crossref PubMed Scopus (61) Google Scholar), have no effect (18Chang C. Snook J.T. The cholesterolaemic effects of dietary fats in cholesteryl ester transfer protein transgenic mice.Br. J. Nutr. 2001; 85: 643-648Crossref PubMed Google Scholar), or increase (15Khosla P. Hajri T. Pronczuk A. Hayes K.C. Replacing dietary palmitic acid with elaidic acid (t-C18:1 delta9) depresses HDL and increases CETP activity in cebus monkeys.J. Nutr. 1997; 127: 531-536Crossref Google Scholar, 24Gupta S.V. Yamada N. Fungwe T.V. Khosla P. Replacing 40% of dietary animal fat with vegetable oil is associated with lower HDL cholesterol and higher cholesterol ester transfer protein in cynomolgus monkeys fed sufficient linoleic acid.J. Nutr. 2003; 133: 2600-2606Crossref PubMed Scopus (6) Google Scholar) plasma CETP activity. These different findings might be attributable to variations in the design of the diets, in which the amount of fat and the presence or absence of cholesterol in the diet was overlooked. We have shown previously that dietary fats modify the regulation of cholesterol 7-α hydroxylase (cyp7) by dietary cholesterol (25Cheema S.K. Cikaluk D. Agellon L.B. Dietary fats modulate the regulatory potential of dietary cholesterol on cholesterol 7 alpha-hydroxylase gene expression.J. Lipid Res. 1997; 38: 315-323Abstract Full Text PDF PubMed Google Scholar). The regulation of cyp7 is similar to that of CETP in that both genes are induced by dietary cholesterol (17Jiang X.C. Agellon L.B. Walsh A. Breslow J.L. Tall A. Dietary cholesterol increases transcription of the human cholesteryl ester transfer protein gene in transgenic mice. Dependence on natural flanking sequences.J. Clin. Invest. 1992; 90: 1290-1295Crossref PubMed Google Scholar, 26Jelinek D.F. Andersson S. Slaughter C.A. Russell D.W. Cloning and regulation of cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme in bile acid biosynthesis.J. Biol. Chem. 1990; 265: 8190-8197Abstract Full Text PDF PubMed Google Scholar). Cholesterol-mediated induction involves the binding of liver X receptor α/retinoid X receptor (LXRα/RXR) to the proximal promoter region of both cyp7 (27Lehmann 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) and CETP (28Luo Y. Tall A.R. Sterol upregulation of human CETP expression in vitro and in transgenic mice by an LXR element.J. Clin. Invest. 2000; 105: 513-520Crossref PubMed Scopus (309) Google Scholar) genes. Fatty acid-mediated induction of the cyp7 gene involves peroxisome proliferator-activated receptor α (PPARα)/RXR, and the peroxisome proliferator response element (PPRE) site is embedded within the LXRα/RXR binding site (29Cheema S.K. Agellon L.B. The murine and human cholesterol 7alpha-hydroxylase gene promoters are differentially responsive to regulation by fatty acids mediated via peroxisome proliferator-activated receptor alpha.J. Biol. Chem. 2000; 275: 12530-12536Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). On the other hand, the regulation of CETP by dietary fats is not clear. It is also not known whether fatty acids interfere with the cholesterol-mediated induction of CETP. In this study, we investigated the regulation of CETP in transgenic mice expressing the human CETP gene under the control of its natural flanking region (CETP-TG mice) by the quantity and quality of dietary fats in the absence or presence of dietary cholesterol. We further investigated whether the quantity and quality of dietary fats alter the expression of LXRα, which in turn might modulate the regulatory potential of cholesterol on CETP expression. We established that a high-fat diet enriched in MUFAs inhibited LXRα expression and that addition of cholesterol to this diet did not rescue the expression of LXRα. We further established that cholesterol added to a high-fat diet enriched in MUFAs suppressed CETP gene expression. Therefore, this study reports for the first time that the quantity and quality of dietary fats inhibit the expression of LXRα, which is responsible for the decreased stimulation of CETP by dietary cholesterol, when given along with high-fat MUFAs. The possibility of regulation of CETP activity by the cross-talk between PPARα and LXRα is discussed. Transgenic mice expressing human CETP under the control of its natural flanking region (3.4 kb) were obtained from Dr. Alan Tall (Columbia University, New York) (17Jiang X.C. Agellon L.B. Walsh A. Breslow J.L. Tall A. Dietary cholesterol increases transcription of the human cholesteryl ester transfer protein gene in transgenic mice. Dependence on natural flanking sequences.J. Clin. Invest. 1992; 90: 1290-1295Crossref PubMed Google Scholar). C57BL6/J female mice were purchased from Jackson Laboratories (Bar Harbor, ME). The transgenic mice were crossbred with C57BL6/J mice to obtain heterozygotes (30Cheema S.K. Rashid-Kolvear F. Streptozotocin-induced increase in cholesterol ester transfer protein (CETP) and its reversal by insulin in transgenic mice expressing human CETP.Can. J. Physiol. Pharmacol. 2003; 81: 997-1004Crossref PubMed Scopus (9) Google Scholar). Animals were housed in a temperature-controlled (25°C) and humidity-controlled room with a 12 h light/dark cycle. Animals were given rodent chow diet and water for ad libitum consumption during the entire period. Eight week old CETP-TG mice were fed a semipurified diet (custom-made basal mix without fat; ICN Biomedicals, Inc., Quebec, Canada) containing either low fat (5%, w/w) or high fat (20%, w/w) from olive oil (enriched in 18:1; a MUFA diet) or beef tallow (enriched in 18:0; a SFA diet) in the presence or absence of 1% cholesterol. Fatty acid analysis of the diets showed that the MUFA diet contains 63% (w/w) 18:1, whereas the SFA diet contains 52% (w/w) 18:0. The animals were fed the specified diets for 2 weeks. Body weight was determined at the beginning and end of the diet study. Food was replenished every other day, and food consumption was recorded. At the end of the diet study, food was withdrawn from all groups 12 h before euthanasia. Mice were euthanized, and blood was collected by cardiac puncture in tubes containing EDTA. Livers were removed, weighed, and quick-frozen in liquid nitrogen. Liver samples were stored at −70°C until further use. All procedures were in accordance with the principles and guidelines of the Canadian Council on Animal Care and the Principles of Laboratory Animal Care (National Institutes of Health) and were approved by the Memorial University of Newfoundland Animal Welfare Committee. Fasting blood was collected via cardiac puncture in a syringe containing 1 g EDTA/l, and plasma was separated. Lipids were extracted from liver samples as described previously (31Yokode M. Hammer R.E. Ishibashi S. Brown M.S. Goldstein J.L. Diet-induced hypercholesterolemia in mice: prevention by overexpression of LDL receptors.Science. 1990; 250: 1273-1275Crossref PubMed Scopus (143) Google Scholar). Plasma and liver lipid samples were assayed for total cholesterol using enzymatic methods (kit 352; Sigma-Aldrich, St. Louis, MO), and triacylglycerol levels were assayed using kit 339 (Sigma-Aldrich). The plasma HDL-cholesterol concentration was determined after precipitating β-lipoproteins with phosphotungstate and measuring cholesterol concentration in the supernatant by enzymatic methods (kit 352; Sigma-Aldrich). The plasma LDL-cholesterol concentration was calculated from plasma total cholesterol, HDL-cholesterol, and triacylglycerol concentrations (32Friedewald W.T. Levy R.I. Fredrickson D.S. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge.Clin. Chem. 1972; 18: 499-502Crossref PubMed Scopus (64) Google Scholar). Plasma CETP activity was assayed using a commercial CETP activity assay kit (Roar Biomedical Research, Inc., Columbia University) to identify transgenic mice expressing human CETP at 5 and 8 weeks of age. Cholesteryl ester transfer activity assays for all other experiments were performed using the radioisotope assay as described previously with modifications (30Cheema S.K. Rashid-Kolvear F. Streptozotocin-induced increase in cholesterol ester transfer protein (CETP) and its reversal by insulin in transgenic mice expressing human CETP.Can. J. Physiol. Pharmacol. 2003; 81: 997-1004Crossref PubMed Scopus (9) Google Scholar). The percentage transfer of radioactivity from HDL to LDL in a control sample was subtracted from that in the test samples, and the values for CETP activity were expressed as percentage cholesteryl ester transfer per hour. CETP mass in plasma samples was quantitatively assayed using the CETP ELISA DAIICHI kit (Daiichi Pure Chemicals Co.) as described previously (30Cheema S.K. Rashid-Kolvear F. Streptozotocin-induced increase in cholesterol ester transfer protein (CETP) and its reversal by insulin in transgenic mice expressing human CETP.Can. J. Physiol. Pharmacol. 2003; 81: 997-1004Crossref PubMed Scopus (9) Google Scholar). Concentrations of the samples were calculated using a standard curve developed using a CETP stock solution of known concentration. To detect changes in CETP mRNA abundance, total RNA from mouse livers was purified according to standard procedures (33Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). Hepatic CETP mRNA levels were determined by reverse transcription and in vitro DNA amplification (30Cheema S.K. Rashid-Kolvear F. Streptozotocin-induced increase in cholesterol ester transfer protein (CETP) and its reversal by insulin in transgenic mice expressing human CETP.Can. J. Physiol. Pharmacol. 2003; 81: 997-1004Crossref PubMed Scopus (9) Google Scholar). The amount of CETP mRNA was normalized to β-actin mRNA content and expressed as arbitrary units. Chimeric gene constructs, harboring serial deletions of the human CETP gene 5′ regulatory region linked to chloramphenicol acetyl transferase (CAT) as a reporter, were designed (34Oliveira H.C. Chouinard R.A. Agellon L.B. Bruce C. Ma L. Walsh A. Breslow J.L. Tall A.R. Human cholesteryl ester transfer protein gene proximal promoter contains dietary cholesterol positive responsive elements and mediates expression in small intestine and periphery while predominant liver and spleen expression is controlled by 5′-distal sequences. Cis-acting sequences mapped in transgenic mice.J. Biol. Chem. 1996; 271: 31831-31838Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) using the published sequence of the human CETP gene (GenBank accession numbers U71187 and M32992). A 1,520 bp PCR fragment containing 360 bp of the 5′ regulatory region, exon 1, intron 1, and exon 2 was generated in which the HindIII site was inserted in exon 1. The PCR product was digested with HindIII, and the resulting fragment containing 300 bp of the CETP gene 5′ regulatory region was linked to CAT in pCAT.Basic vector (Promega) and designated 300CETP.CAT. Fragment 138CETP.CAT was constructed by digesting 300CETP.CAT with XbaI. For the longer CETP gene 5′ regulatory region, the 1,520 bp PCR fragment was used to screen a human chromosome 16 genomic library. An XbaI-XbaI fragment containing sequences from −3,420 to −138 was obtained and cloned into 138CETP.CAT to generate 3400CETP.CAT. 3400CETP.CAT was digested with KpnI to generate 570CETP.CAT (Fig. 1). The sequences of all cloned CETP fragments were confirmed, and restriction mapping of 3400CETP.CAT, 570CETP.CAT, and 138CETP.CAT chimeric gene constructs was performed to confirm the identity of the plasmids. SW 872 cells (human liposarcoma cells) were purchased from the American Tissue Culture Collection (Manassas, VA). The cells were standardized to grow in a humidified atmosphere of 5% CO2/95% air in DMEM/F12 (3:1), 5% FBS, 10 mM HEPES, and 50 μg/ml gentamycin at 37°C according to previously published methods (35Richardson M.A. Berg D.T. Johnston P.A. McClure D. Grinnell B.W. Human liposarcoma cell line, SW872, secretes cholesteryl ester transfer protein in response to cholesterol.J. Lipid Res. 1996; 37: 1162-1166Abstract Full Text PDF PubMed Google Scholar). When cells reached 70% confluence, they were differentiated as described below. Briefly, medium was removed and replaced by DMEM/F12 (3:1), 10 mM HEPES, 50 μg/ml gentamycin, 100 μg/ml oleic acid complexed to fatty acid-free BSA, 1 μg/ml insulin, and 1 μg/ml transferrin for 24 h. After 24 h, cells were washed with PBS (0.14 M NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4.7H2O, and 1.5 mM KH2PO4, pH 7.4) and fresh regular growth medium (without serum) containing DMEM/F12 (3:1), 10 mM HEPES, and 50 μg/ml gentamycin was added. Cells were grown under these conditions for 24 h before transfection. To investigate the effect of fatty acids and cholesterol on CETP gene regulation, the CETP chimeric gene constructs (138CETP.CAT, 570CETP.CAT, and 3400CETP.CAT) were transfected into SW 872 cells according to the CaPO4 transfection method (29Cheema S.K. Agellon L.B. The murine and human cholesterol 7alpha-hydroxylase gene promoters are differentially responsive to regulation by fatty acids mediated via peroxisome proliferator-activated receptor alpha.J. Biol. Chem. 2000; 275: 12530-12536Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The total amount of plasmid DNA was kept constant by adding sonicated salmon sperm DNA. The cells were cotransfected with β-galactosidase to control for transfection efficiency. Regular growth medium containing delipidated serum (2.5% FBS) was added to the transfected cells. Oleic acid (a monounsaturated fatty acid; 18:1) was complexed to fatty acid free-BSA (29Cheema S.K. Agellon L.B. The murine and human cholesterol 7alpha-hydroxylase gene promoters are differentially responsive to regulation by fatty acids mediated via peroxisome proliferator-activated receptor alpha.J. Biol. Chem. 2000; 275: 12530-12536Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), and 25-OH cholesterol (Sigma-Aldrich) was dissolved in DMSO (Sigma-Aldrich) and added to the transfected cells (50 μM for fatty acids and 4 μg/ml for 25-OH cholesterol). Control cells received the vehicle alone. After 24 h, cells were washed twice with PBS, replenished with regular growth medium containing delipidated serum (5% FBS), oleic acid, or 25-OH cholesterol for an additional 24 h. Cells were harveste
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