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Genetic control of HDL levels and composition in an interspecific mouse cross (CAST/Ei × C57BL/6J)

2000; Elsevier BV; Volume: 41; Issue: 12 Linguagem: Inglês

10.1016/s0022-2275(20)32354-3

1539-7262

Margarete Mehrabian, Lawrence W. Castellani, Ping-Zi Wen, Jack Wong, Tat Rithaporn, Susan Hama, G P Hough, David Johnson, John J. Albers, Giuliano A. Mottino, Joy S. Frank, Mohamad Navab, Alan M. Fogelman, Aldons J. Lusis,

Peroxisome Proliferator-Activated Receptors

Strain CAST/Ei (CAST) mice exhibit unusually low levels of high density lipoproteins (HDL) as compared with most other strains of mice, including C57BL/6J (B6). This appears to be due in part to a functional deficiency of lecithin:cholesterol acyltransferase (LCAT). LCAT mRNA expression in CAST mice is normal, but the mice exhibit several characteristics consistent with functional deficiency. First, the activity and mass of LCAT in plasma and in HDL of CAST mice were reduced significantly. Second, the HDL of CAST mice were relatively poor in phospholipids and cholesteryl esters, but rich in free cholesterol and apolipoprotein A-I (apoA-I). Third, the adrenals of CAST mice were depleted of cholesteryl esters, a phenotype similar to that observed in LCAT- and acyl-CoA:cholesterol acyltransferase-deficient mice. Fourth, in common with LCAT-deficient mice, CAST mice contained triglyceride-rich lipoproteins with "panhandle"-like protrusions. To examine the genetic bases of these differences, we studied HDL lipid levels in an intercross between strain CAST and the common laboratory strain B6 on a low fat, chow diet as well as a high fat, atherogenic diet. HDL levels exhibited complex inheritance, as 12 quantitative trait loci with significant or suggestive likelihood of observed data scores were identified. Several of the loci occurred over plausible candidate genes and these were investigated. The results indicate that the functional LCAT deficiency is unlikely to be due to variations of the LCAT gene. Our results suggest that novel genes are likely to be important in the control of HDL metabolism, and they provide evidence of genetic factors influencing the interaction of LCAT with HDL.—Mehrabian, M., L. W. Castellani, P-Z. Wen, J. Wong, T. Rithaporn, S. Y. Hama, G. P. Hough, D. Johnson, J. J. Albers, G. A. Mottino, J. S. Frank, M. Navab, A. M. Fogelman, and A. J. Lusis. Genetic control of HDL levels and composition in an interspecific mouse cross (CAST/Ei × C57BL/6J). J. Lipid Res. 2000. 41: 1936–1946. Strain CAST/Ei (CAST) mice exhibit unusually low levels of high density lipoproteins (HDL) as compared with most other strains of mice, including C57BL/6J (B6). This appears to be due in part to a functional deficiency of lecithin:cholesterol acyltransferase (LCAT). LCAT mRNA expression in CAST mice is normal, but the mice exhibit several characteristics consistent with functional deficiency. First, the activity and mass of LCAT in plasma and in HDL of CAST mice were reduced significantly. Second, the HDL of CAST mice were relatively poor in phospholipids and cholesteryl esters, but rich in free cholesterol and apolipoprotein A-I (apoA-I). Third, the adrenals of CAST mice were depleted of cholesteryl esters, a phenotype similar to that observed in LCAT- and acyl-CoA:cholesterol acyltransferase-deficient mice. Fourth, in common with LCAT-deficient mice, CAST mice contained triglyceride-rich lipoproteins with "panhandle"-like protrusions. To examine the genetic bases of these differences, we studied HDL lipid levels in an intercross between strain CAST and the common laboratory strain B6 on a low fat, chow diet as well as a high fat, atherogenic diet. HDL levels exhibited complex inheritance, as 12 quantitative trait loci with significant or suggestive likelihood of observed data scores were identified. Several of the loci occurred over plausible candidate genes and these were investigated. The results indicate that the functional LCAT deficiency is unlikely to be due to variations of the LCAT gene. Our results suggest that novel genes are likely to be important in the control of HDL metabolism, and they provide evidence of genetic factors influencing the interaction of LCAT with HDL.—Mehrabian, M., L. W. Castellani, P-Z. Wen, J. Wong, T. Rithaporn, S. Y. Hama, G. P. Hough, D. Johnson, J. J. Albers, G. A. Mottino, J. S. Frank, M. Navab, A. M. Fogelman, and A. J. Lusis. Genetic control of HDL levels and composition in an interspecific mouse cross (CAST/Ei × C57BL/6J). J. Lipid Res. 2000. 41: 1936–1946. The inverse relationship between high density lipoprotein (HDL) levels and coronary artery disease (CAD) (1Rhoads G.G. Gulbrandsen C.L. Kugan A. Serum lipoproteins and coronary artery disease in a population study of Hawaii Japanese men.N. Engl. J. Med. 1976; 294: 293-298Google Scholar) has generated interest in the metabolism of HDL and the environmental and genetic factors contributing to variations in HDL levels. The major structural proteins of HDL are apolipoprotein A-I (apoA-I) and apoA-II, but HDL also contains numerous other proteins, including lecithin: cholesterol acyl-transferase (LCAT), serum paraoxonase (PON1), platelet-activating factor acetylhydrolase (PAF-AH), apoE, apoA-IV, cholesteryl ester transfer protein (CETP), and phospholipid transfer protein (PLTP). HDL are extremely heterogeneous, and various species of HDL differ in functions relevant to CAD, such as the ability to promote cholesterol efflux and to inhibit LDL oxidation (2Castellani L.W. Navab M. Van Lenten B.J. Hedrick C.C Hama S.Y. Goto A.M. Fogelman A.M. Lusis A.J. Overexpression of apolipoprotein AII in transgenic mice converts high density lipoproteins to proinflammatory particles.J. Clin. Invest. 1997; 100: 464-474Google Scholar). Biochemical and physiologic studies have revealed that HDL are derived from the transfer of surface components of triglyceride-rich lipoproteins into HDL (3Havel R.J. Kane J.P. Kashyae M.L. Interchange of apolipoproteins between chylomicrons and high density lipoproteins during alimentary lipenia in man.J. Clin. Invest. 1973; 52: 32-38Google Scholar, 4Tall A.R. Green P.H.R. Glickman R.M. Riley J.W. Metabolic fate of chylomicron phospholipids and apoproteins in the rat.J. Clin. Invest. 1979; 64: 977-989Google Scholar) and from the association of cellular plasma membranes with apoA-I (3Havel R.J. Kane J.P. Kashyae M.L. Interchange of apolipoproteins between chylomicrons and high density lipoproteins during alimentary lipenia in man.J. Clin. Invest. 1973; 52: 32-38Google Scholar, 5Hamilton R.H. Williams M.C. Fielding C.J. Havel R.J. Discoidal bilayer structure of nascent high density lipoproteins from perfused rat liver.J. Clin. Invest. 1976; 58: 667-680Google Scholar). Studies of human mutations and transgenic/knockout mice have clarified the functions of various HDL proteins in vivo. Human metabolic studies have also indicated that common variations in HDL cholesterol levels are strongly related to the fractional catabolic rate of apoA-I (6Brinton E.A. Eisenberg S. Breslow J.L. Human HDL cholesterol levels are determined by apoA-1 fractional catabolic rate, which correlates inversely with estimates of HDL particle size. Effects of gender, hepatic and lipoprotein lipases, triglyceride and insulin levels, and body fat distribution.Arterioscler. Thromb. 1994; 14: 707-720Google Scholar). However, the genetic factors contributing to common variation in HDL levels and functional differences among human populations are poorly understood. One significant genetic determinant of HDL levels is a polymorphism of the promoter region of hepatic lipase, which contributes to HDL levels in males (7Cohen J.C. Vega G.L. Grundy S.M. Hepatic lipase: new insights from genetic and metabolic studies.Curr. Opin. Lipidol. 1999; 10: 259-267Google Scholar). The identification of the ATPase-binding cassette transporter responsible for Tangier disease has revealed a novel pathway for HDL metabolism, and some evidence suggests that variations of the transporter could contribute to common HDL deficiencies (8Bodzioch M. Orso E. Klucken J. Langmann T. Böttcher A. Diederich W. Drobnik W. Barlage S. Büchler C. Porsch-Özcürümez M. Kaminski W.E. Hahmann H.W. Oette K. Rothe G. Aslanidis C. Lackner K.J. Schmitz G. The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease.Nat. Genet. 1999; 22: 347-351Google Scholar, 9Brooks-Wilson A. Marcil M. Clee S.M. Zhang L-H. Roomp K. van Dam M. Yu L. Brewer C. Collins J.A. Molhuizen O.F. Loubser O. Ouelette B.F.F. Fichter K. Ashbourne-Excoffon K.J.D. Sensen C.W. Scherer S. Mott S. Denis M. Martindale D. Frohlich J. Morgan K. Koop B. Pimstone S. Kastelein J.J.P. Genest Jr., J. Hayden M.R. Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency.Nat. Genet. 1999; 22: 336-345Google Scholar, 10Rust S. Rosier M. Funke H. Real J. Amoura Z. Piette J.C. Deleuze J.F. Brewer H.B. Duverger N. Denefle P. Assmann G. Tangier disease is caused by mutations in the gene encoding ATP-binding cassette transporter 1.Nat. Genet. 1999; 22: 352-355Google Scholar). We now report studies of the genetic control of HDL levels in a mouse animal model. The mouse has become increasingly useful for the study of complex traits. As compared with human studies, the use of animal models reduces the effects of environmental variables and greatly simplifies linkage analysis (11Lander E.S. Schork N.J. Genetic dissection of complex traits.Science. 1994; 265: 2037-2048Google Scholar). Common laboratory inbred strains of mice exhibit variations in HDL levels, functions, and structures (12Lusis A.J. Taylor B.A. Wangenstein R.W. LeBoeuf R.C. Genetic control of lipid transport in mice. II. Genes controlling structure of high density lipoproteins.J. Biol. Chem. 1983; 258: 5071-5078Google Scholar), and we have previously reported biochemical and genetic studies of some of these variations (13Paigen B. Mitchell D. Reue K. Morrow A. Lusis A.J. LeBoeuf R.C. Ath-1, a gene determining atherosclerosis susceptibility and high density lipoprotein levels in mice.Proc. Natl. Acad. Sci. USA. 1987; 84: 3763-3767Google Scholar, 14Doolittle M.H. LeBoeuf R.C. Warden C.H. Bee L.M. Lusis A.J. A polymorphism affecting apolipoprotein A-II translational efficiency determines high density lipoprotein size and composition.J. Biol. Chem. 1990; 265: 16380-16388Google Scholar, 15Mehrabian M. Qiao J-H. Hyman R.H. Ruddle D. Laughton C. Lusis A.J. Influence of the apoA-II gene locus on HDL levels and fatty streak development in mice.Arterioscler. Thromb. 1993; 13: 1-10Google Scholar, 16Purcell-Huynh D.A. Weinreb A. Castellani L.W. Mehrabian M. Doolittle M.H. Lusis A.J. Genetic factors in lipoprotein metabolism: analysis of a genetic cross between inbred mouse strains NZB/BINJ and SM/J using a complete linkage map approach.J. Clin. Invest. 1995; 96: 1845-1858Google Scholar, 17Warden C.H. Fisler J.S. Shoemaker S.M. Wen P-Z. Svenson K.L. Pace M.J. Lusis A.J. Identification of four chromosomal loci determining obesity in a multifactorial mouse model.J. Clin. Invest. 1995; 95: 1545-1552Google Scholar, 18Shih D.M. Gu L. Hama S. Xia Y. Navab M. Fogelman A.M. Lusis A.J. Genetic-dietary regulation of serum paraoxonase expression and its role in atherogenesis in a mouse model.J. Clin. Invest. 1996; 97: 1630-1639Google Scholar, 19Welch C.L. Xia Y-R. Shechter I. Farese R. Mehrabian M. Mehdizadeh S. Warden C.H. Lusis A.J. Genetic regulation of cholesterol homeostasis: chromosomal organization of candidate genes.J. Lipid Res. 1996; 37: 1406-1421Google Scholar, 20Machleder D. Ivandic B. Welch C.L. Castellani L. Reue K. Lusis A.J. Complex genetic control of HDL levels in mice in response to an atherogenic diet: coordinate regulation of HDL levels and bile acid metabolism.J. Clin. Invest. 1997; 99: 1406-1419Google Scholar, 21Mehrabian M. Wen P-Z. Fisler J. Davis R.C. Lusis A.J. Genetic loci controlling body fat, lipoprotein metabolism, and insulin levels in a multifactorial mouse model.J. Clin. Invest. 1998; 101: 2485-2496Google Scholar). In the present study, we have examined the genetic control of HDL levels in an intercross between the laboratory strain C57BL/6J (B6) and the distantly related (∼1 million years) strain CAST/Ei (CAST), which is derived from a separate subspecies of mouse. These studies were performed with two different diets, one a low fat, chow diet and the other a high fat, atherogenic diet, to examine genetic-dietary interactions. The results have revealed a complex pattern of inheritance, with more than a dozen loci contributing to HDL levels and compositions on the two diets. Candidate genes at some of the loci were examined for possible involvement. The results suggest that a significant proportion of the genetic component in HDL metabolism is unlikely to be explained by the usual candidates for plasma lipid metabolism. They also reveal that CAST mice have low HDL levels due, in part, to reduced LCAT activity, and that this, in turn, results in a form of adrenal lipid depletion. Interestingly, the low LCAT activity is not due to reduced LCAT expression, but, apparently, to a genetic factor influencing the interaction of LCAT with HDL. All mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and were housed under conditions meeting Association for Assessment and Accreditation of Laboratory Animal Care. CAST males were mated with B6 females and the resulting F1 progeny were intercrossed to produce the F2 progeny. The F2 mice were weaned at about 21 days of age onto rodent chow containing 6% of calories from fat (Purina 5001) and at about 4 months of age, they were switched to a high fat, high cholesterol diet for 5 weeks. This diet was 75% chow supplemented with 7.5% cocoa butter (resulting in 15% of calories from fat) and also 2.5% dextrose, 1.625% each of sucrose and dextrin, 1.25% cholesterol, and 0.05% sodium cholate (Diet No. 90221; Harlan Teklad, Madison, WI). The mice were given ad libitum access to food and were maintained on a 12-h light-dark cycle throughout. Briefly, mice were fasted overnight before collection of blood and were killed ∼3 h into the diurnal phase of the light cycle. The kidneys, liver, and adrenals were collected for DNA isolation and other analyses. Plasma lipoproteins were separated, and lipids were quantitated using enzymatic procedures as previously described (15Mehrabian M. Qiao J-H. Hyman R.H. Ruddle D. Laughton C. Lusis A.J. Influence of the apoA-II gene locus on HDL levels and fatty streak development in mice.Arterioscler. Thromb. 1993; 13: 1-10Google Scholar). LCAT activity and PLTP activity in plasma were determined as previously described (22Miyazaki A. Rahim A.T. Ohta T. Morino Y. Horiuchi S. High density lipoprotein mediates selective reduction in cholesteryl esters from macrophage foam cells.Biochim. Biophys. Acta. 1992; 1126: 73-80Google Scholar, 23Cheung M.C. Wolfbauer G. Albers J.J. Plasma phospholipid mass transfer rate: relationship to plasma phospholipid and cholesteryl ester transfer activities and lipid parameters.Biochim. Biophys. Acta. 1996; 1303: 103-110Google Scholar). Mouse lipoproteins were isolated either by density ultracentrifugation or by fast performance liquid chromatography (FPLC) (24Hedrick C.C. Castellani L.W. Warden C.H. Puppione D.L. Lusis A.J. Influence of mouse apolipoprotein A-II on plasma lipoproteins in transgenic mice.J. Biol. Chem. 1993; 268: 676-682Google Scholar). For FPLC, 450 μl of pooled heparinized plasma was injected onto two Pharmacia (Uppsala, Sweden) Superose 6 columns connected in series, and lipoproteins eluted with 154 mM NaCl and 3 mM sodium azide in endotoxin-free water, pH 8.2. Fractions of 1.0 ml each were collected. The lipoprotein elution profile was determined by measuring cholesterol, and appropriate fractions were pooled. Cholesterol was determined by an enzymatic assay as described (15Mehrabian M. Qiao J-H. Hyman R.H. Ruddle D. Laughton C. Lusis A.J. Influence of the apoA-II gene locus on HDL levels and fatty streak development in mice.Arterioscler. Thromb. 1993; 13: 1-10Google Scholar). The pooled lipoproteins were filtered through molecular weight cutoff (CUFC4) filters to remove the sodium azide present in the FPLC effluent (Millipore, Boston, MA). Mouse liver membranes were prepared by homogenizing 1 g of liver in 10 ml of 150 mM NaCl, 1 mM CaCl2, 10 mM Tris-HCl (pH 7.5), 0.5 μM leupeptin, 1 mM phenylmethylsulfonyl fluoride, pH 7.5 (buffer A). A low speed supernatant was first prepared by centrifuging the homogenate at 500 g for 5 min and then at 8,000 g for 15 min. The supernatant was then spun at 100,000 g (24,000 rpm in an SW40Ti rotor) for 1 h and the supernatant was discarded. The pellet was suspended in 1 ml of buffer A by flushing 10 times through a 1-ml syringe with a 22-gauge needle. The dispersed pellet was transferred to a new ultracentrifuge tube, 4 ml of buffer A was added, and the mixture was spun at 100,000 g for 60 min. The supernatant was discarded and the pellet was resuspended in 0.5 ml of 50 mM NaCl, 1 mM CaCl2, and 20 mM Tris-HCl, pH 7.5. Protein concentrations were determined by the Bio-Rad (Hercules, CA) dye-binding protein assay. Protein electrophoresis was carried out using Novex (San Diego, CA) NuPAGE 4–12% precast polyacrylamide gradient gels either under reducing or nonreducing conditions as described by Novex. Proteins were transferred onto nitrocellulose membranes and visualized by enhanced chemiluminescence (ECL) detection (Amersham, Little Chalfont, Buckinghamshire, UK). Quantification of the specific luminescent protein bands was performed with Image-Quant software (Molecular Dynamics, Sunnyvale, CA). For scavenger receptor class B type I (SR-BI) immunoblotting, 10 μg of solubilized liver membranes was subjected to electrophoresis and transferred to filters, and incubated overnight with a 1:2,000 dilution of mouse SR-BI antibody (provided by M. C. deBeer, University of Kentucky, Lexington, KY). For LCAT immunoblotting, a 1:500 dilution of human LCAT antibody (Data Medical Associates, Arlington, TX) was used to detect LCAT protein in either mouse plasma (2 μl), isolated HDL prepared by density ultracentrifugation (0.5 μg total protein), or FPLC fractions (10 μl). Antibodies against mouse apoA-I or apoE were purchased from Biodesign International (Kennebunk, ME) and used at dilutions of 1:5,000 and 1:1,000, respectively. For apoD immunoblotting, a 1:500 dilution of human apoD antibody (Cortex Biochem, San Leandro, CA) was used to detect apoD in 1–3 μl of mouse plasma. Total RNA was isolated from liver according to the Trizol reagent protocol (Gibco, Life Technologies, Grand Island, NY). Northern blot analysis was used to quantitate the mRNA levels of LCAT. For each sample, 15 μg of RNA was electrophoresed through formaldehyde-1% agarose gels and transferred to 20 × SSC-equilibrated Hybond ECL nitrocellulose membranes. Membranes were hybridized after UV cross-linking and washed at a high stringency (65°C, 0.1× SSC). Northern blots were probed with a 32P-labeled mouse LCAT polymerase chain reaction (PCR) product. The forward primer was 5′-GGGTGCTGTTGCTGT TGG-3′; and the reverse primer was 5′-TGCCAAAGCCAGGGA CAC-3′. The blots were also hybridized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA to normalize the quantities of RNA loaded onto the gel lanes. A complete linkage map for all chromosomes except the Y chromosome was constructed with microsatellite markers and restriction fragment length variants as previously described (21Mehrabian M. Wen P-Z. Fisler J. Davis R.C. Lusis A.J. Genetic loci controlling body fat, lipoprotein metabolism, and insulin levels in a multifactorial mouse model.J. Clin. Invest. 1998; 101: 2485-2496Google Scholar). PCR primers for microsatellite typing were purchased from Research Genetics (Huntsville, AL). Linkage maps were constructed with the Map Manager version 2.6.5 (25Manly K.F. A Macintosh program for storage and analysis of experimental genetic mapping data.Mamm. Genome. 1993; 4: 303-313Google Scholar) program. Statistical comparison of quantitative traits between groups, as shown in the tables, was performed by analysis of variance and regression (Statview 4.5; Abacus Concepts, Berkeley, CA). Likelihood of the observed data (LOD) scores for quantitative traits were calculated with the MAPMAKER/QTL program (26Lander E.S. Green P. Abrahamson J. Barlow A. Daley M. Lincoln S. Newburg L. MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations.Genomics. 1987; 1: 174-181Google Scholar). The data were adjusted for the effects of age and sex by regression. Negatively stained lipid preparations were made by placing a drop of lipid solution onto a copper grid (300 mesh). Uranyl acetate stain was gently added to the drop and incubated for 3–5 min. After this time excess fluid was wicked off the grid, which was allowed to dry and then viewed in a JEOL (Tokyo, Japan) 100CX electron microscope. Two samples from CAST and B6 on both a chow and high fat diet were prepared. Fifty different grid squares for each sample were examined. cDNA was prepared from total liver RNA of CAST and B6 mice, using random hexamers. Overlapping PCR primers were designed using OLIGO, a primer analysis program by Molecular Biology Insights (Cascade, CO; http://www.oligo.net). Fragments were then PCR amplified in 1.5 mM MgCl2 buffer, using a "touchdown" protocol with temperatures ranging from 68 to 50°C. The products were purified and sequenced with an Applied Biosystems (Foster City, CA) automated sequencer. The results were analyzed by several sequencing programs: BLAST and Entrez at http://www.ncbi.nlm.nih.gov; CAP3, a sequencing assembly programming algorithm at http://gcg.tigem.it/ASSEMBLY/assemble.html; and Genedoc at http://www.cris.com/~ketchup/genedoc.shtml. The translations into six reading frames were performed with the Baylor College of Medicine (Houston, TX) sequence analysis programs at their Website http://dot.imgen.bcm.tmc.edu:9331/seq-util/seq-util.html. Among various strains of mice surveyed (12Lusis A.J. Taylor B.A. Wangenstein R.W. LeBoeuf R.C. Genetic control of lipid transport in mice. II. Genes controlling structure of high density lipoproteins.J. Biol. Chem. 1983; 258: 5071-5078Google Scholar), CAST are exceptional in their low levels of HDL cholesterol on a chow diet as well as a high fat, atherogenic diet containing cholic acid (Table 1). Most strains of mice, such as BALB/c and C3H, maintain relatively high levels of HDL cholesterol (70–90 mg/dl) on an atherogenic diet whereas CAST mice and B6 mice exhibit about a 2-fold decrease (Table 1). We have previously proposed that the decrease in HDL cholesterol in strain C57BL/6J mice in response to the atherogenic diet is related to decreased expression of cholesterol 7α-hydroxylase (CYP7A1) (20Machleder D. Ivandic B. Welch C.L. Castellani L. Reue K. Lusis A.J. Complex genetic control of HDL levels in mice in response to an atherogenic diet: coordinate regulation of HDL levels and bile acid metabolism.J. Clin. Invest. 1997; 99: 1406-1419Google Scholar). The results with CAST mice are consistent with this, because CAST mice also exhibited a decrease in CYP7A1 mRNA levels (Table 1). The FPLC lipoprotein profile of CAST mice is shown in Fig. 1. In addition to these differences in HDL levels, CAST mice were unusually responsive to an atherogenic diet in terms of the levels of low density lipoprotein/very high density lipoprotein (LDL/VLDL) cholesterol (Fig. 1). In genetic studies, described below, HDL levels did not cosegregate with LDL/VLDL levels, indicating that the low HDL and high LDL/VLDL in CAST mice result, at least in part, from independent genetic factors. The composition of CAST HDL was also distinctive as compared with common laboratory strains of mice, including B6. After isolation by sequential density gradient ultracentrifugation, the HDL from CAST mice were determined to have relatively low levels of phospholipids and cholesteryl esters but high levels of free cholesterol (Fig. 2, Table 1).TABLE 1.Comparison of HDL levels and related parameters in CAST and B6 mice on two dietsChow Diet (mean ± SE)Atherogenic Diet (mean ± SE)TraitB6CASTB6CASTHDL cholesterol (mg/dl)54 ± 741± 2*30 ± 218 ± 2*Paraoxonase (activity)82 ± 1660 ± 943 ± 319 ± 2*LCAT mRNA (relative units)5.5 ± 0.74.4 ± 0.84.1 ± 0.33.7 ± 0.5LCAT activity (units)48.6 ± 0.331.3 ± 0.2*20.2 ± 0.21.0 ± 0.1*PAF-AH activity (units)6.3 ± 0.36.2 ± 0.76.9 ± 0.26.4 ± 0.3PLTP activity (units)10.4 ± 1.32.9 ± 0.3*11.6 ± 2.64.8 ± 0.4*Plasma cholesterol (mg/dl)68 ± 663 ± 2125 ± 15764 ± 18*Plasma triglyceride (mg/dl)123 ± 1049 ± 9*65 ± 9244 ± 32*Plasma apoAI (mg/dl)119 ± 798 ± 898 ± 1899 ±13Plasma UC/TC0.14 ± 0.010.37 ± 0.02*0.17 ± 0.010.28 ± 0.0*HDL UC/TC0.22 ± 0.010.25 ± 0.010.20 ± 0.010.30 ± 0.01CYP7A1 mRNA (relative units)1.3 ± 0.32.0 ± 0.60.4 ± 0.11.2 ± 0.1SR-BI mRNA (relative units)1.5 ± 0.20.9 ± 0.1*3.3 ± 0.01.9 ± 0.0*SR-BI protein (relative units)1.8 ± 0.11.7 ± 0.21.2 ± 0.01.0 ± 0.0Abbreviations: HDL, high density lipoprotein; UC, unesterified cholesterol; TC, total cholesterol; CYP7A1, cytochrome P450 7A (cholesterol 7α-hydroxylase).Female mice 4–6 months of age were studied on a chow diet or after feeding an atherogenic diet for 5 weeks. Values represent the means of three to seven independent measurements. Levels significantly different (P < 0.05) between CAST and B6 mice are indicated with an asterisk(*). Open table in a new tab Fig. 2.High density lipoprotein (HDL) from CAST mice exhibit reduced levels of total phospholipids. HDL were ultracentrifugally isolated from CAST and B6 mice, maintained on either chow or atherogenic diets, and analyzed by positive ion mass spectrometry for the presence of certain phospholipids. A total of 25 μg of HDL protein was injected in each experiment. The native phospholipid species analyzed in the HDL were lysophosphatidylcholine (lysoPC; m/z = 496.2), palmitoyl linolyl phosphatidylcholine (PLPC; m/z = 758.3), and palmitoyl arachidonoyl phosphatidylcholine (PAPC; m/z = 782.3). Chow diet: lysoPC, P ⩽ 0.05; PLPC, P ⩽ 0.05; PAPC, P < 0.001. High fat atherogenic diet (Ath): lysoPC, P < 0.0001; PLPC, P < 0.001, PAPC, P < 0.001. Three or more preparations were analyzed per group.View Large Image Figure ViewerDownload (PPT) Abbreviations: HDL, high density lipoprotein; UC, unesterified cholesterol; TC, total cholesterol; CYP7A1, cytochrome P450 7A (cholesterol 7α-hydroxylase). Female mice 4–6 months of age were studied on a chow diet or after feeding an atherogenic diet for 5 weeks. Values represent the means of three to seven independent measurements. Levels significantly different (P < 0.05) between CAST and B6 mice are indicated with an asterisk(*). The unusual composition and low levels of HDL cholesterol in CAST mice suggested that they may exhibit a deficiency of LCAT. Therefore, we examined LCAT activities in plasmas of CAST and B6 mice maintained on both chow and atherogenic diets. CAST mice exhibited decreased levels, particularly on the atherogenic diet (Fig. 3A). To test whether the decreased activity was due to a reduced mass or a structural mutation of LCAT affecting activity, LCAT mass was quantitated with a monospecific antibody. The amount of LCAT detected in plasma by Western analysis was similar in CAST and B6 mice on both diets (Fig. 3B). Moreover, both strains exhibited similar levels of hepatic LCAT mRNA on both diets, indicating that a promoter variation of the LCAT gene is not involved (Fig. 3C). One potential mechanism for the reduced LCAT activity is that the LCAT binds poorly to HDL, resulting in increased enzyme turnover and HDL depleted in cholesteryl esters. To examine this possibility we examined LCAT protein in HDL isolated by ultracentrifugation. In B6 mice, most of the plasma LCAT was associated with the ultracentrifugally isolated HDL, whereas in CAST mice, significantly lower levels were observed (Fig. 3B). Because the isolation of HDL by ultracentrifugation subjects the particles to high salt and pressure, we also examined the association of LCAT with HDL after FPLC analysis in 0.15 M NaCl. Under those low salt conditions, the levels of LCAT activity and mass associated with HDL were similar in CAST and B6 mice (data not shown). Thus, CAST LCAT appears to be associated with HDL under physiologic conditions, but is dissociated in part under the conditions of ultracentrifugation. To examine the possibility that structural differences in LCAT between the two strains contributed to a loss in high salt, hepatic LCAT cDNA was sequenced to check for possible mutations that could influence the function of the protein (Table 2). For this, hepatic mRNA was reverse transcribed and subjected to PCR amplification of specific regions of the LCAT-coding sequence. Two nucleotide changes were identified, but both of these resulted in relatively conservative amino acid changes, L19→V and M412→L. These data suggest that the deficiency in LCAT activity is most likely due to altered interaction of LCAT with HDL or other lipoproteins (see Discussion) in CAST mice. It is possible that this results from the above-described missense variations, although this seems unlikely given the conservative nature of the substitutions and the genetic data discussed below. Alternatively, CAST HDL or plasma may contain a factor or factors influencing the interaction of LCAT with HDL.TABLE 2.Sequence comparison of LCAT cDNA from CAST and B6 miceaLiver mRNA for LCAT was reverse transcribed and amplified from CAST and B6 mice and sequenced in both directions. Differences in the sequence are indicated in bold face and the corresponding amino acids are shown.Nucleotide and Amino Acid NumberMouse (B6)Mouse (CAST)RatHumanNucleotide 63CTGGTGCTGCTGAmino acid 19LVLLNucleotide 1242ATGCTGCTGCTGAmino acid 412MLLLa Liver mRNA for LCAT was reverse transcribed and amplified from CAST and B6 mice and sequenced in both directions. Differences in the sequence are indicated in bold face and the corresponding amino acids are shown. Open table in a new tab Because of the unusually low levels of HD