Induction of Fatal Inflammation in LDL Receptor and ApoA-I Double-Knockout Mice Fed Dietary Fat and Cholesterol
2003; Elsevier BV; Volume: 163; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)63480-3
ISSN1525-2191
AutoresManal Zabalawi, Shaila Bhat, Tara Loughlin, Michael J. Thomas, Eric T. Alexander, Mark Cline, Bill C. Bullock, Mark C. Willingham, Mary G. Sorci‐Thomas,
Tópico(s)Adipokines, Inflammation, and Metabolic Diseases
ResumoAtherogenic response to dietary fat and cholesterol challenge was evaluated in mice lacking both the LDL receptor (LDLr−/−) and apoA-I (apoA-I−/−) gene, LDLr−/−/apoA-I−/− or double-knockout mice. Gender- and age-matched LDLr−/−/apoA-I−/− mice were fed a diet consisting of 0.1% cholesterol and 10% palm oil for 16 weeks and compared to LDLr−/− mice or single-knockout mice. The LDLr−/− mice showed a 6- to 7-fold increase in total plasma cholesterol (TPC) compared to their chow-fed mice counterparts, while LDLr−/−/apoA-I−/− mice showed only a 2- to 3-fold increase in TPC compared to their chow-fed controls. This differential response to the atherogenic diet was unanticipated, since chow-fed LDLr−/− and LDLr−/−/apoA-I−/− mice began the study with similar LDL levels and differed primarily in their HDL concentration. The 6-fold diet-induced increase in TPC observed in the LDLr−/− mice occurred mainly in VLDL/LDL and not in HDL. Mid-study plasma samples taken after 8 weeks of diet feeding showed that LDLr−/− mice had TPC concentrations approximately 60% of their 16-week level, while the LDLr−/−/apoA-I−/− mice had reached 100% of their 16-week TPC concentration after only 8 weeks of diet. Male LDLr−/− mice showed similar aortic cholesterol levels to male LDLr−/−/apoA-I−/− mice despite a 4-fold higher VLDL/LDL concentration in the LDLr−/− mice. A direct comparison of the severity of aortic atherosclerosis between female LDLr−/− and LDLr−/−/apoA-I−/− mice was compromised due to the loss of female LDLr−/−/apoA-I−/− mice between 10 and 14 weeks into the study. Diet-fed female and, with time, male LDLr−/−/apoA-I−/− mice suffered from severe ulcerated cutaneous xanthomatosis. This condition, combined with a complete depletion of adrenal cholesterol, manifested in fatal wasting of the affected mice. In conclusion, LDLr−/− and LDLr−/−/apoA-I−/− mice showed dramatic TPC differences in response to dietary fat and cholesterol challenge, while despite these differences both genotypes accumulated similar levels of aortic cholesterol. Atherogenic response to dietary fat and cholesterol challenge was evaluated in mice lacking both the LDL receptor (LDLr−/−) and apoA-I (apoA-I−/−) gene, LDLr−/−/apoA-I−/− or double-knockout mice. Gender- and age-matched LDLr−/−/apoA-I−/− mice were fed a diet consisting of 0.1% cholesterol and 10% palm oil for 16 weeks and compared to LDLr−/− mice or single-knockout mice. The LDLr−/− mice showed a 6- to 7-fold increase in total plasma cholesterol (TPC) compared to their chow-fed mice counterparts, while LDLr−/−/apoA-I−/− mice showed only a 2- to 3-fold increase in TPC compared to their chow-fed controls. This differential response to the atherogenic diet was unanticipated, since chow-fed LDLr−/− and LDLr−/−/apoA-I−/− mice began the study with similar LDL levels and differed primarily in their HDL concentration. The 6-fold diet-induced increase in TPC observed in the LDLr−/− mice occurred mainly in VLDL/LDL and not in HDL. Mid-study plasma samples taken after 8 weeks of diet feeding showed that LDLr−/− mice had TPC concentrations approximately 60% of their 16-week level, while the LDLr−/−/apoA-I−/− mice had reached 100% of their 16-week TPC concentration after only 8 weeks of diet. Male LDLr−/− mice showed similar aortic cholesterol levels to male LDLr−/−/apoA-I−/− mice despite a 4-fold higher VLDL/LDL concentration in the LDLr−/− mice. A direct comparison of the severity of aortic atherosclerosis between female LDLr−/− and LDLr−/−/apoA-I−/− mice was compromised due to the loss of female LDLr−/−/apoA-I−/− mice between 10 and 14 weeks into the study. Diet-fed female and, with time, male LDLr−/−/apoA-I−/− mice suffered from severe ulcerated cutaneous xanthomatosis. This condition, combined with a complete depletion of adrenal cholesterol, manifested in fatal wasting of the affected mice. In conclusion, LDLr−/− and LDLr−/−/apoA-I−/− mice showed dramatic TPC differences in response to dietary fat and cholesterol challenge, while despite these differences both genotypes accumulated similar levels of aortic cholesterol. The plasma concentration of high-density lipoproteins (HDL) and apolipoprotein A-I (apoA-I) are considered to be reliable measures of an individual's risk of developing coronary heart disease (CHD).1Boden WE High-density lipoprotein cholesterol as an independent risk factor in cardiovascular disease: assessing the data from Framingham to the Veterans Affairs high-density lipoprotein intervention trial.Am J Cardiol. 2000; 86: 19L-22LAbstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar Analysis of four of the largest human epidemiology studies suggest that for every 1 mg/dl increase in HDL apoA-I, a 2% decrease in CHD risk for men and a 3% decrease for women may result.2Gordon DJ Probstfield JL Garrison RJ Neaton JD Castelli WP Knoke JD Jacobs Jr, DR Bangdiwala S Tyroler HA High-density lipoprotein cholesterol and cardiovascular disease: four prospective american studies.Circulation. 1989; 79: 8-15Crossref PubMed Scopus (2679) Google Scholar Additionally, the use of animal models has established that inhibition or regression of atherosclerosis is highly dependent on HDL apoA-I concentrations,3Navab M Van Lenten BJ Reddy ST Fogelman AM High-density lipoprotein and the dynamics of atherosclerotic lesions.Circulation. 2001; 104: 2386-2387Crossref PubMed Scopus (29) Google Scholar demonstrated by the use of transgenic mice and rabbits,4Walsh A Ito Y Breslow JL High levels of human apolipoprotein A-I in transgenic mice result in increased plasma levels of small high-density lipoprotein (HDL) particles comparable to human HDL3.J Biol Chem. 1989; 264: 6488-6494Abstract Full Text PDF PubMed Google Scholar, 5Rubin EM Ishida BY Clift SM Krauss RM Expression of human apolipoprotein A-I in transgenic mice results in reduced plasma levels of murine apolipoprotein A-I and the appearance of two new high-density lipoprotein size subclasses.Proc Natl Acad Sci USA. 1991; 88: 434-438Crossref PubMed Scopus (242) Google Scholar, 6Duverger N Viglietta C Berthou L Emmanuel F Tailleux A Parmentier-Nihoul L Laine B Fievet C Castro G Fruchart JC Houbebine LM Denèfle P Transgenic rabbits expressing human apolipoprotein A-I in the liver.Arterioscler Thromb Vasc Biol. 1996; 16: 1424-1429Crossref PubMed Scopus (55) Google Scholar and by repeated intravenous administration of apoA-I.7Badimon JJ Badimon L Fuster V Regression of atherosclerotic lesions by high-density lipoprotein plasma fraction in the cholesterol-fed rabbit.J Clin Invest. 1990; 85: 1234-1241Crossref PubMed Scopus (676) Google Scholar, 8Miyazaki A Sakuma S Morikawa W Takiue T Miake F Terano T Sakai M Hakamata H Sakamoto YI Naito M Ruan YM Takahashi K Ohta T Horiuchi S Intravenous injection of rabbit apolipoprotein A-I inhibits the progression of atherosclerosis in cholesterol-fed rabbits.Arterioscler Thromb Vasc Biol. 1995; 15: 1882-1888Crossref PubMed Scopus (167) Google Scholar, 9Chiesa G Monteggia E Marchesi M Lorenzon P Laucello M Lorusso V Di Mario C Karvouni E Newton RS Bisgaier CL Franceschini G Sirtori CR Recombinant apolipoprotein A-IMilano infusion into rabbit carotid artery rapidly removes lipid from fatty streaks.Circ Res. 2002; 90: 974-980Crossref PubMed Scopus (174) Google Scholar A more complicated picture of the role of HDL apoA-I in CHD prevention has emerged from studies in mice with targeted deletions of the mouse apoA-I gene,10Williamson R Lee D Hagaman J Maeda N Marked reduction of high-density lipoprotein cholesterol in mice genetically modified to lack apolipoprotein A-I.Proc Natl Acad Sci USA. 1992; 89: 7134-7138Crossref PubMed Scopus (190) Google Scholar, 11Li H Reddick RL Maeda N Lack of apoA-I is not associated with increased susceptibility to atherosclerosis in mice.Arterioscler Thromb. 1993; 13: 1814-1821Crossref PubMed Scopus (163) Google Scholar because the mere absence of plasma apoA-I does not lead to a greater atherosclerosis susceptibility in mice. Rather, it appears from studies of transgenic mice with targeted deletions,12Hughes SD Verstuyft J Rubin EM HDL deficiency in genetically engineered mice requires elevated LDL to accelerate atherogenesis.Arterioscler Thromb Vasc Biol. 1997; 17: 1725-1729Crossref PubMed Scopus (46) Google Scholar, 13Voyiaziakis E Goldberg IJ Plump AS Rubin EM Breslow JL Huang LS ApoA-I deficiency causes both hypertriglyceridemia and increased atherosclerosis in human apoB transgenic mice.J Lipid Res. 1998; 39: 313-321Abstract Full Text Full Text PDF PubMed Google Scholar, 14Liu AC Lawn RM Verstuyft JG Rubin EM Human apolipoprotein A-I prevents atherosclerosis associated with apolipoprotein[a] in transgenic mice.J Lipid Res. 1994; 35: 2263-2267Abstract Full Text PDF PubMed Google Scholar or from studies of somatic gene transfer15Kawashiri MA Zhang Y Pure E Rader DJ Combined effects of cholesterol reduction and apolipoprotein A-I expression on atherosclerosis in LDL receptor deficient mice.Atherosclerosis. 2002; 165: 15-22Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 16De Geest B Zhao Z Collen D Holvoet P Effects of adenovirus-mediated human apoA-I gene transfer on neointima formation after endothelial denudation in apoE-deficient mice.Circulation. 1997; 43: 4349-4356Crossref Scopus (90) Google Scholar, 17Tangirala RK Praticó D FitzGerald GA Chun S Tsukamoto K Maugeais C Usher DC Puré E Rader DJ Reduction of isoprostanes and regression of advanced atherosclerosis by apolipoprotein.Eur J Biol Chem. 2001; 276: 261-266Crossref PubMed Scopus (105) Google Scholar, 18Rong JX Li J Reis ED Choudhury RP Dansky HM Elmalem VI Fallon JT Breslow JL Fisher EA Elevating high-density lipoprotein cholesterol in apolipoprotein E-deficient mice remodels advanced atherosclerotic lesions by decreasing macrophage and increasing smooth muscle cell content.Circulation. 2001; 104: 2447-2452Crossref PubMed Scopus (192) Google Scholar, 19Benoit P Emmanuel F Caillaud JM Bassinet L Castro G Gallix P Fruchart JC Branellec D Denèfle P Duverger N Somatic gene transfer of human apoA-I inhibits atherosclerosis progression in mouse models.Circulation. 1999; 99: 105-110Crossref PubMed Scopus (133) Google Scholar and from studies using combinations of targeted disruptions, as in apoE and apoA-I knockout mice,20Plump AS Scott CJ Breslow JL Human apolipoprotein A-I gene expression increases high density lipoprotein and suppresses atherosclerosis in the apolipoprotein E-deficient mouse.Proc Natl Acad Sci USA. 1994; 91: 9607-9611Crossref PubMed Scopus (546) Google Scholar, 21Pászty C Maeda N Verstuyft J Rubin EM Apolipoprotein AI transgene corrects apolipoprotein E deficiency-induced atherosclerosis in mice.J Clin Invest. 1994; 94: 899-903Crossref PubMed Scopus (340) Google Scholar the development of atherosclerosis depends on the type and amount of atherogenic particles in plasma as well as the concentration of HDL apoA-I. Thus, it appears that HDL apoA-I protects the vascular wall in situations in which concentrations of VLDL and LDL could potentially cause development of atherosclerosis. Although this conclusion has not been completely supported by studies in humans who either lack plasma apoA-I or possess apoA-I mutations that lead to very low plasma concentrations of HDL apoA-I,22Sorci-Thomas MG Thomas MJ The effects of altered apolipoprotein A-I structure on plasma HDL concentration.Trends Cardiovasc Med. 2002; 12: 121-128Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar it is likely that populations of individuals with apoA-I mutations are too small to provide reliable statistical analysis. Therefore, we undertook the present studies to determine the protective effect of HDL apoA-I concentration in the presence of VLDL and LDL concentrations resembling that seen in LDL receptor deficiency in humans. LDL receptor deficient mice23Ishibashi S Brown MS Goldstein JL Gerard RD Hammer RE Herz J Hypercholesterolemia in low-density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery.J Clin Invest. 1993; 92: 883-893Crossref PubMed Scopus (1281) Google Scholar, 24Ishibashi S Goldstein JL Brown MS Herz J Burns DK Massive xanthomatosis and atherosclerosis in cholesterol-fed low-density lipoprotein receptor-negative mice.J Clin Invest. 1994; 93: 1885-1893Crossref PubMed Scopus (602) Google Scholar and apoA-I deficient mice10Williamson R Lee D Hagaman J Maeda N Marked reduction of high-density lipoprotein cholesterol in mice genetically modified to lack apolipoprotein A-I.Proc Natl Acad Sci USA. 1992; 89: 7134-7138Crossref PubMed Scopus (190) Google Scholar, 11Li H Reddick RL Maeda N Lack of apoA-I is not associated with increased susceptibility to atherosclerosis in mice.Arterioscler Thromb. 1993; 13: 1814-1821Crossref PubMed Scopus (163) Google Scholar were crossed to obtain double-knockout mice, LDLr−/−/apoA-I−/−. These mice were fed a chow or an atherogenic diet and the effects on plasma lipoproteins and atherosclerosis were examined. LDL receptor knockout (LDLr−/−) mice and apoA-I knockout (apoA-I−/−) were obtained from Jackson Lab (Bar Harbor, ME) where both lines had been bred six to eight times into the C57BL/6 background. LDLr−/− mice were fully crossed into apoA-I−/− mice, to obtain LDLr−/−/apoA-I−/− mice. All mice were housed at the Wake Forest University Baptist Medical Center where procedures were approved by the Animal Care and Use Committee of the Wake Forest University Health Sciences. Experimental mice were weaned at 21 days and fed a chow diet (Purina). At 30 days of age blood was obtained by orbital sinus bleeding after a 4- to 6-hour fast for baseline evaluation. The animals were anesthetized using a 1:1 mixture of ketamine (50 mg/ml) and xylazine (10 mg/ml) in which 1 μl per gram body weight was injected intramuscularly behind the knee. The orbital sinus bleeding was performed by inserting a micro hematocrit capillary tube into the retro-orbital plexus. Up to 250 μl of whole blood was drawn. The animal's eye was treated with an opthalamic ointment (Lacri-Lube) to keep the eyes lubricated and the mouse was then placed on a 37°C heating pad. The respiration, orientation, and balance of the mouse was monitored for at least 2 hours. At 6 weeks of age, a group of mice were fed an atherogenic diet containing 10% saturated fat from palm oil and 0.1% cholesterol for 16 weeks, as previously described.25Rudel LL Kelley K Sawyer JK Shah R Wilson MD Dietary monounsaturated fatty acids promote aortic atherosclerosis in LDL receptor - null, human apoB100 - overexpression transgenic mice.Arterioscler Thromb Vasc Biol. 1998; 18: 1818-1827Crossref PubMed Scopus (155) Google Scholar, 26Furbee JW Sawyer JK Parks JS Lecithin: cholesterol acyltransferase (LCAT) deficiency increases atherosclerosis in the low-density lipoprotein receptor (LDLr) or apolipoprotein E (apoE) knockout mice.J Biol Chem. 2002; 277: 3511-3519Crossref PubMed Scopus (82) Google Scholar As controls, both gender- and age-matched mice of each genotype were fed a Purina chow diet for 16 weeks. All mice were maintained in a temperature-controlled room with a 12-hour light and 12-hour dark cycle. At the end of the diet period both chow- and diet-fed mice were fasted then anesthetized as described above. Blood was obtained by cardiac puncture. Approximately 1 ml of blood was collected in a 1.5-ml tube containing 20 μl of 0.5 μmol/L ethylenediaminetetraacetate (EDTA) pH 8.0/1 μmol/L sodium azide and placed immediately on ice. The blood was centrifuged immediately at 4°C at 10,000 rpm for 10 minutes. The plasma was removed and stored at −20°C. The animal was opened via midline laparotomy to expose the thoracic and abdominal cavities. The heart and aorta were removed and suspended in 10% formalin for at least 48 hours before further analysis. For all other tissues, sections were cut and stored in 10% formalin. For chemical analysis additional sections of tissue were frozen in liquid nitrogen and stored at −80°C for later use. Plasma from both chow- and diet-fed mice was analyzed for total plasma, free and esterified cholesterol and triglycerides by enzymatic assay (Roche Molecular Biochemical, Indianapolis, IN). HDL cholesterol was determined after dextran-sulfate precipitation of plasma as described previously.27Warnick GR Dextran sulfate-Mg2+ precipitation procedure for quantification of high-density lipoprotein cholesterol.Clin Chem. 1982; 28: 1379-1388Crossref PubMed Scopus (1814) Google Scholar, 28Sorci-Thomas MG Thomas M Curtiss L Landrum M Single repeat deletion in apoA-I blocks cholesterol esterification and results in rapid catabolism of Δ6 and wild-type apoA-I in transgenic mice.J Biol Chem. 2000; 275: 12156-12163Crossref PubMed Scopus (43) Google Scholar The plasma lipoprotein distribution was determined for chow- and diet-fed mice using fast protein liquid chromatography (FPLC). Approximately 200 μl of plasma from chow-fed and 100 μl from diet-fed mice were applied to two Superose-6 columns connected in tandem and run at 0.5 ml/min in a buffer containing 0.9% NaCl, 0.26 mmol/L EDTA (pH 7.4), and 1.5 mmol/L NaN3. Approximately 75,500-μl fractions were collected and 50 to 100 μl from each fraction was used for lipid determination, as previously described.28Sorci-Thomas MG Thomas M Curtiss L Landrum M Single repeat deletion in apoA-I blocks cholesterol esterification and results in rapid catabolism of Δ6 and wild-type apoA-I in transgenic mice.J Biol Chem. 2000; 275: 12156-12163Crossref PubMed Scopus (43) Google Scholar To assess the apoprotein distribution, 10 μl from chow or 20 μl from diet-fed FPLC-fractionated plasma were separated on a 13% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electrotransferred to nitrocelluose at 4°C for 1 hour at 1 Amp using 25 mmol/L Tris pH 8.0 192 mmol/L glycine and 20% methanol. Western analysis was carried as previously described28Sorci-Thomas MG Thomas M Curtiss L Landrum M Single repeat deletion in apoA-I blocks cholesterol esterification and results in rapid catabolism of Δ6 and wild-type apoA-I in transgenic mice.J Biol Chem. 2000; 275: 12156-12163Crossref PubMed Scopus (43) Google Scholar using antibodies raised against mouse apoA-I or mouse apoE (Biodesign, Saco, ME). To assess the apoprotein distribution in mouse plasma from LDLr−/− and LDLr−/−/apoA-I−/− mice, freshly drawn plasma was subjected to density-gradient ultracentrifugation. Approximately 250 μl of plasma was adjusted to 1.27 g/ml using KBr, then overlayered with 12 to 13 ml of a 1.25 g/ml KBr solution. The tubes were sealed, placed in a pre-cooled Ti 70.1 rotor and centrifuged at 4°C, 50,000 rpm for 18 hours. The top 3 ml from each centrifuge tube was exhaustively dialyzed against 10 mmol/L ammonium bicarbonate (pH 7.4), 3 μmol/L EDTA, and 15 μmol/L sodium azide. Following dialysis, the protein concentration was determined by Lowry assay.29Lowry OJ Rosebrough NJ Farr AL Randall RJ Protein measurement with the Folin phenol reagent.J Biol Chem. 1951; 19: 265-275Abstract Full Text PDF Google Scholar Aliquots containing 10 μg of protein were concentrated and separated by SDS-PAGE on 4% to 30% gradient gels. Each lane contained approximately 10 μg of protein and was compared to the Mark 12 molecular weight standard (Invitrogen, Carlsbad, CA). Gels were stained with Coomassie brilliant blue, destained and their image recorded using an α Innotech Imaging system. Atherosclerosis evaluations were carried out as previously described.25Rudel LL Kelley K Sawyer JK Shah R Wilson MD Dietary monounsaturated fatty acids promote aortic atherosclerosis in LDL receptor - null, human apoB100 - overexpression transgenic mice.Arterioscler Thromb Vasc Biol. 1998; 18: 1818-1827Crossref PubMed Scopus (155) Google Scholar, 26Furbee JW Sawyer JK Parks JS Lecithin: cholesterol acyltransferase (LCAT) deficiency increases atherosclerosis in the low-density lipoprotein receptor (LDLr) or apolipoprotein E (apoE) knockout mice.J Biol Chem. 2002; 277: 3511-3519Crossref PubMed Scopus (82) Google Scholar Briefly, the heart and aorta were placed under a dissecting microscope and the adventitia was completely removed. Then the aorta was detached at the base of the heart, blotted, weighed and placed in a glass tube with 3 ml of chloroform-methanol 2:1 (v/v), and 5α-cholestane as the internal standard. Extracted lipid was dried under a stream of N2 at 60°C to remove solvent. The lipid was dissolved in hexane and injected onto a ZB-50 gas-liquid chromatography (GLC) column for free cholesterol determination. The remaining lipid extract was dried again, dissolved in 1 ml of ethanol and 0.1 ml 50% KOH was added to each tube. The mixture was then heated at 60°C for 1 hour. Following the incubation, 0.5 ml of hexane and 1 ml of water was added, the tubes were centrifuged to separate the phases. The top phase was transferred and injected onto a DB17 GLC column for total cholesterol measurement. Esterified cholesterol was calculated as the difference between free and total cholesterol. Finally, delipidated aortic tissue was digested and dissolved in 1N NaOH and the total protein content determined by Lowry.29Lowry OJ Rosebrough NJ Farr AL Randall RJ Protein measurement with the Folin phenol reagent.J Biol Chem. 1951; 19: 265-275Abstract Full Text PDF Google Scholar Adrenal glands from both chow- and diet-fed mice were taken at the time of necropsy, weighed and then placed under a dissecting microscope. The surrounding fat was removed before GLC analysis using the same method as described above for the quantification of cholesterol in the mouse aorta.25Rudel LL Kelley K Sawyer JK Shah R Wilson MD Dietary monounsaturated fatty acids promote aortic atherosclerosis in LDL receptor - null, human apoB100 - overexpression transgenic mice.Arterioscler Thromb Vasc Biol. 1998; 18: 1818-1827Crossref PubMed Scopus (155) Google Scholar, 26Furbee JW Sawyer JK Parks JS Lecithin: cholesterol acyltransferase (LCAT) deficiency increases atherosclerosis in the low-density lipoprotein receptor (LDLr) or apolipoprotein E (apoE) knockout mice.J Biol Chem. 2002; 277: 3511-3519Crossref PubMed Scopus (82) Google Scholar Sections of skin were taken from the back and abdomen at the time of necropsy and frozen. At the time of analysis, 200 to 400 mg of tissue were subjected to lipid extraction and aliquots were taken, dried, and re-suspended in water and 0.5% Triton-X100. Enzymatic analysis was performed on aliquots to determine free and total cholesterol content. All samples were compared to cholesterol standards of known concentration, which had been treated in an analogous manner. Sections of liver, skin, and adrenal gland from chow- and diet-fed mice were fixed with 10% formalin at the end of the study. These tissues were embedded in paraffin, sectioned, and then stained with eosin and counterstained with hematoxylin. The slides were examined under the Zeiss Axioplan 2 microscope and digital images recorded. Images were then prepared for publication using Adobe Photoshop 6.0. Data are presented as the mean ± SEM or ± SD as indicated. Data were analyzed by one-way analysis of variance using StatView 5.0.1 and then individual differences between pairing of groups were found using Fisher's least significant post hoc test. Statistical significance was considered at P ≤ 0.05. This study was initiated to determine the effects of a dietary fat and cholesterol challenge on LDLr−/−/apoA-I−/− mice. All mice were age- and gender-matched and fed either chow or an atherogenic diet, comprised of 0.1% cholesterol and 10% palm oil, for 16 weeks. After 8 weeks of consuming the atherogenic diet the mice were bled, and plasma lipid determinations were conducted. After 16 weeks of diet-feeding, all mice were euthanized and complete plasma lipoprotein and atherosclerosis analyses were conducted. Table 1 shows the plasma lipid values for chow- and diet-fed LDLr−/− or single-knockout mice and LDLr−/−/apoA-I−/− or double-knockout mice after 8 and 16 weeks of diet-feeding. Chow-fed LDLr−/− and LDLr−/−/apoA-I−/− mice showed distinctly different TPC values which ranged between 200 to 350 mg/dl, with no significant differences between males and females within a genotype. After consuming the atherogenic diet for 8 weeks the LDLr−/− mice showed TPC levels that were approximately 60% to 70% of their 16-week level, reaching 1482 ± 166 mg/dl, while after 8 weeks of consuming the diet, LDLr−/−/apoA-I−/− mice reached their maximum TPC concentration of 700 ± 37 mg/dl (Table 1). The significant difference between the two genotypes was that LDLr−/− mice showed a 7-fold increase in TPC compared to their chow-fed counterparts following 16 weeks of diet, while LDLr−/−/apoA-I−/− mice showed only a modest 3-fold increase in TPC after 16 weeks of diet, with neither genotype showing gender-related differences (Table 1).Table 1Plasma Lipid Levels in LDLr−/− and LDLr−/−/Apo A-I−/− MiceGenotype (n)GenderTPC (mg/dl)FC (mg/dl)EC (mg/dl)TG (mg/dl)FC/TC16 weeks of chow LDLr−/− (5)Female370 ± 43a98 ± 10a271 ± 32a75 ± 4a0.31 ± 0.02a LDLr−/− (4)Male302 ± 18a82 ± 22a220 ± 17a109 ± 13b0.32 ± 0.01a LDLr−/−/apo A–I−/− (5)Female192 ± 15b74 ± 16a118 ± 15b101 ± 16b0.45 ± 0.03b LDLr−/−/apo A–I−/− (5)Male212 ± 24b80 ± 12a127 ± 28b152 ± 11c0.47 ± 0.03b8 weeks of diet LDLr−/− (5)Female1577 ± 121c444 ± 27b1133 ± 113c202 ± 39c0.29 ± 0.022c LDLr−/− (4)Male1388 ± 211c376 ± 42b1011 ± 171c109 ± 24b0.28 ± 0.020c LDLr−/−/apo A–I−/− (7)Female630 ± 33d202 ± 6c427 ± 28d90 ± 17a,b0.36 ± 0.012d LDLr−/−/apo A–I−/− (17)Male748 ± 42d245 ± 12c488 ± 39d109 ± 24b0.37 ± 0.009d16 weeks of diet LDLr−/− (5)Female2341 ± 112c555 ± 17d1768 ± 95e214 ± 18c0.24 ± 0.008e LDLr−/− (4)Male2226 ± 108e520 ± 20d1705 ± 9e136 ± 15c0.23 ± 0.005e LDLr−/−/apo A–I−/− (7)Female546 ± 50d187 ± 13c359 ± 30d264 ± 19d0.37 ± 0.001d LDLr−/−/apo A–I−/− (17)Male652 ± 22d197 ± 15c456 ± 20d188 ± 15f0.33 ± 0.009aBlood samples were collected after either a 4- to 6-hour fast. Plasma lipid values were determined by enzymatic assay as described in Experimental Procedures. All values represent the mean ± SEM, n = number of animals in the indicated group. Lowercase letters indicate significant differences at P < 0.05. Open table in a new tab Blood samples were collected after either a 4- to 6-hour fast. Plasma lipid values were determined by enzymatic assay as described in Experimental Procedures. All values represent the mean ± SEM, n = number of animals in the indicated group. Lowercase letters indicate significant differences at P < 0.05. In contrast to plasma cholesterol levels, 8 weeks triglyceride values for LDLr−/− mice were 155 ± 29 mg/dl and increased slightly to 200 ± 15 mg/d by 16 weeks (Table 1), while triglycerides in LDLr−/−/apoA-I−/− mice steadily increased from their 8-week value of 98 ± 20 mg/dl to 220 ± 17 mg/dl by 16 weeks (Table 1). At 16 weeks of diet, female LDLr−/−/apoA-I−/− mice showed a 1.4-fold higher triglyceride level than male LDLr−/−/apoA-I−/− mice (Table 1). Both the type of diet and duration of the feeding affected the plasma ratio of free and total cholesterol in LDLr−/− and LDLr−/−/apoA-I−/− mice in this study. Chow-fed LDLr−/− mice had an FC/TC ratio of 0.31 ± 0.02, while LDLr−/−/apoA-I−/− mice averaged a FC/TC ratio of 0.46 ± 0.03. The increase in FC relative to TC strongly suggests that the absence of plasma apoA-I in the LDLr−/−/apoA-I−/− mice resulted in impairment of LCAT-mediated cholesterol esterification. Reduced LCAT activation yields less plasma ester cholesterol (EC) and the reduction in plasma EC is not apparently overcome by the presence of other plasma apoproteins that might activate LCAT (apoE, apoAIV, and apoCs), suggesting that in mice, apoA-I assumes a major role in regulating plasma EC concentrations.22Sorci-Thomas MG Thomas MJ The effects of altered apolipoprotein A-I structure on plasma HDL concentration.Trends Cardiovasc Med. 2002; 12: 121-128Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 28Sorci-Thomas MG Thomas M Curtiss L Landrum M Single repeat deletion in apoA-I blocks cholesterol esterification and results in rapid catabolism of Δ6 and wild-type apoA-I in transgenic mice.J Biol Chem. 2000; 275: 12156-12163Crossref PubMed Scopus (43) Google Scholar Supporting this idea, a report in apoA-I−/− mice10Williamson R Lee D Hagaman J Maeda N Marked reduction of high-density lipoprotein cholesterol in mice genetically modified to lack apolipoprotein A-I.Proc Natl Acad Sci USA. 1992; 89: 7134-7138Crossref PubMed Scopus (190) Google Scholar, 30Parks JS Li H Gebre AK Smith TL Maeda N Effect of apolipoprotein A-I deficiency on lecithin: cholesterol acyltransferase activation in mouse plasma.J Lipid Res. 1995; 36: 349-355Abstract Full Text PDF PubMed Google Scholar showed that the relative increase in the free cholesterol/total cholesterol (FC/TC) ratio or the concomitant decrease in the EC/TC ratio is associated with a lack of plasma apoA-I, since control mice expressing both apoA-I+/+ alleles displayed an EC/TC ratio of 0.60, and mice lacking both apoA-I−/− alleles showed an EC/TC ratio of 0.38. The FC/TC ratio was also affected by the length of time the mice were fed an atherogenic diet. When mice of either genotype were fed for 8 weeks, the FC/TC ratio was significantly reduced from the chow-fed control level, as seen in Table 1. This trend continued, and by 16 weeks the FC/TC ratio had dropped approximately 28% in LDLr−/− mice and by 24% in LDLr−/−/apoA-I−/− mice, when compared to their chow-fed counterparts. It is possible that this decrease reflects an accumulation of VLDL and LDL CE since these particles are not cleared via the LDL receptor23Ishibashi S Brown MS Goldstein JL Gerard RD Hammer RE Herz J Hypercholesterolemia in low-density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gen
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