ABCA1 plays no role in the centripetal movement of cholesterol from peripheral tissues to the liver and intestine in the mouse
2009; Elsevier BV; Volume: 50; Issue: 7 Linguagem: Inglês
10.1194/jlr.m900024-jlr200
ISSN1539-7262
AutoresChonglun Xie, Stephen D. Turley, John M. Dietschy,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoThis study uses the mouse to explore the role of ABCA1 in the movement of this cholesterol from the peripheral organs to the endocrine glands for hormone synthesis and liver for excretion. The sterol pool in all peripheral organs was constant and equaled 2,218 and 2,269 mg/kg, respectively, in abca1+/+ and abca1−/− mice. Flux of cholesterol from these tissues equaled the rate of synthesis plus the rate of LDL-cholesterol uptake and was 49.9 mg/day/kg in control animals and 62.0 mg/day/kg in abca1−/− mice. In the abca1+/+ animals, this amount of cholesterol moved from HDL into the liver for excretion. In the abca1−/− mice, the cholesterol from the periphery also reached the liver but did not use HDL. Fecal excretion of cholesterol was just as high in abac1−/− mice (198 mg/day/kg) as in the abac1+/+ animals (163 mg/day/kg), although the abac1−/− mice excreted relatively more neutral than acidic sterols. This study established that ABCA1 plays essentially no role in the turnover of cholesterol in peripheral organs or in the centripetal movement of this sterol to the endocrine glands, liver, and intestinal tract for excretion. This study uses the mouse to explore the role of ABCA1 in the movement of this cholesterol from the peripheral organs to the endocrine glands for hormone synthesis and liver for excretion. The sterol pool in all peripheral organs was constant and equaled 2,218 and 2,269 mg/kg, respectively, in abca1+/+ and abca1−/− mice. Flux of cholesterol from these tissues equaled the rate of synthesis plus the rate of LDL-cholesterol uptake and was 49.9 mg/day/kg in control animals and 62.0 mg/day/kg in abca1−/− mice. In the abca1+/+ animals, this amount of cholesterol moved from HDL into the liver for excretion. In the abca1−/− mice, the cholesterol from the periphery also reached the liver but did not use HDL. Fecal excretion of cholesterol was just as high in abac1−/− mice (198 mg/day/kg) as in the abac1+/+ animals (163 mg/day/kg), although the abac1−/− mice excreted relatively more neutral than acidic sterols. This study established that ABCA1 plays essentially no role in the turnover of cholesterol in peripheral organs or in the centripetal movement of this sterol to the endocrine glands, liver, and intestinal tract for excretion. Every cell in the body is surrounded by a plasma membrane of unique physicochemical characteristics. This membrane is essentially a bilayer of various phospholipids, sphingolipids, and glycolipids interdigitated with unesterified cholesterol molecules. The rigid, hydrophobic ring structure of the sterol interacts through hydrophobic bonding with the fatty acid chains of the phospholipids to condense the bilayer and establish a very hydrophobic region ∼3 nm thick that runs through the center of the membrane (1Simons K. Ikonen E. How cells handle cholesterol.Science. 2000; 290: 1721-1726Crossref PubMed Scopus (1060) Google Scholar, 2Alberts B. Bray D. Lewis J. Raff M. Roberts K. Watson J.D. The plasma membrane.in: Robertson M. Molecular Biology of the Cell. Garland Publishing, New York1989: 275-340Google Scholar). Thus, the typical cell membrane is highly permeable to water but essentially impermeable to protons, electrolytes, and hydrophilic molecules. Also imbedded in the membrane is a variety of signaling and transport proteins that allow the cell to maintain, in a highly selective manner, the metabolic processes essential for life. These functions are all critically dependent upon the presence of the cholesterol molecule in the bulk phase of the plasma membrane. As a consequence, rates of cholesterol synthesis are very high in the growing organs of the developing fetus and newborn animal (3Dietschy J.M. Kita T. Suckling K.E. Goldstein J.L. Brown M.S. Cholesterol synthesis in vivo and in vitro in the WHHL rabbit, an animal with defective low density lipoprotein receptors.J. Lipid Res. 1983; 24: 469-480Abstract Full Text PDF PubMed Google Scholar, 4Belknap W.M. Dietschy J.M. Sterol synthesis and low density lipoprotein clearance in vivo in the pregnant rat, placenta, and fetus.J. Clin. Invest. 1988; 82: 2077-2085Crossref PubMed Scopus (94) Google Scholar–5Quan G. Xie C. Dietschy J.M. Turley S.D. Ontogenesis and regulation of cholesterol metabolism in the central nervous system of the mouse.Brain Res. Dev. Brain Res. 2003; 146: 87-98Crossref PubMed Scopus (129) Google Scholar). Any mutation that limits the availability of this critical molecule during development leads either to intrauterine death of the embryo or to major developmental abnormalities in the newborn (6Salen G. Shefer S. Batta A.K. Tint G.S. Xu G. Honda A. Irons M. Elias E.R. Abnormal cholesterol biosynthesis in the Smith-Lemli-Opitz syndrome.J. 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This renewal process is apparently so important that virtually every cell in the body has invested in the complex biochemical machinery necessary to convert acetyl-CoA into cholesterol (9Turley S.D. Spady D.K. Dietschy J.M. Identification of a metabolic difference accounting for the hyper- and hyporesponder phenotypes of cynomolgus monkey.J. Lipid Res. 1997; 38: 1598-1611Abstract Full Text PDF PubMed Google Scholar, 10Li H. Turley S.D. Liu B. Repa J.J. Dietschy J.M. GM2/GD2 and GM3 gangliosides have no effect on cellular cholesterol pools or turnover in normal or NPC1 mice.J. Lipid Res. 2008; 49: 1816-1828Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Lesser amounts of sterol are also acquired through the receptor-mediated and bulk phase uptake of lipoproteins like LDL (11Liu B. Xie C. Richardson J.A. Turley S.D. Dietschy J.M. Receptor-mediated and bulk-phase endocytosis cause macrophage and cholesterol accumulation in Niemann-Pick C disease.J. 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Central nervous system: cholesterol turnover, brain development and neurodegeneration.Biol. Chem. 2009; (In press.)Crossref PubMed Scopus (220) Google Scholar). Some insights into the molecular events controlling this turnover process came from studies in patients with Tangier disease, a genetic illness characterized by a marked reduction in the level of circulating apolipoprotein AI (apoAI) and cholesterol carried in HDL (HDL-C) and by tissue infiltration of lipid-laden macrophages (14Assmann G. Simantke O. Schaefer H-E. Smootz E. Characterization of high density lipoproteins in patients heterozygous for Tangier disease.J. Clin. Invest. 1977; 60: 1025-1035Crossref PubMed Scopus (40) Google Scholar). With the discovery that this disease was apparently caused by mutations in the transport protein ABCA1 (15Brooks-Wilson A. Marcil M. Clee S.M. Zhang L-H. Roomp K. van Dam M. Yu L. Brewer C. Collins J.A. 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Invest. 1999; 104: R25-R31Crossref PubMed Scopus (649) Google Scholar), a fairly straightforward paradigm was developed to describe the putative events characterizing so-called "reverse cholesterol transport." In this scheme, ABCA1 was envisioned as reducing the activation energy for the desorption of cholesterol from the outer leaflet of the plasma membrane by transferring the sterol, along with phospholipids, directly onto apoAI-rich, nascent HDL particles circulating in the pericellular fluid. These nascent particles would then mature in the circulation and, ultimately, deliver their cargo of cholesterol to the endocrine tissues and liver using pathways mediated by scavenger receptor class B type I, cholesteryl ester transfer protein (CETP), and the LDL receptor (LDLR) (19Tall A.R. Wang N. Mucksavage P. Is it time to modify the reverse cholesterol transport model?.J. Clin. Invest. 2001; 108: 1273-1275Crossref PubMed Scopus (69) Google Scholar, 20Groen A.K. Elferink R.P.J.O. Verkade H.J. Kuipers F. The ins and outs of reverse cholesterol transport.Ann. Med. 2004; 36: 135-145Crossref PubMed Scopus (55) Google Scholar–21Attie A.D. Kastelein J.P. Hayden M.R. Pivotal role of ABCA1 in reverse cholesterol transport influencing HDL levels and susceptibility to atherosclerosis.J. Lipid Res. 2001; 42: 1717-1726Abstract Full Text Full Text PDF PubMed Google Scholar). Importantly, this model implied that plasma membrane cholesterol turnover was driven by events external to the cell and that the velocity of this process would be proportional to the levels of both ABCA1 in the membrane and apoAI in the pericellular fluid. If true, in the absence of ABCA1 activity, there should be sterol accumulation and downregulation of cholesterol synthesis and cholesterol carried in LDL (LDL-C) uptake in all peripheral organs, decreased flux of cholesterol from the periphery to the endocrine glands and liver, and, finally, decreased biliary and fecal sterol excretion. While this was an attractive hypothesis for explaining cholesterol turnover, recent experimental measurements have yielded results contrary to these predictions. For example, in the abca1−/− mouse, an animal model manifesting the same abnormalities in apoAI and HDL-C metabolism as seen in Tangier patients (22McNeish J. Aiello R.J. Guyot D. Turi T. Gabel C. Aldinger C. Hoppe K.L. Roach M.L. Royer L.J. de Wet J. et al.High density lipoprotein deficiency and foam cell accumulation in mice with targeted disruption of ATP-binding cassette transporter-1.Proc. Natl. Acad. Sci. USA. 2000; 97: 4245-4250Crossref PubMed Scopus (481) Google Scholar, 23Christiansen-Weber T.A. Voland J.R. Wu Y. Ngo K. Roland B.L. Nguyen S. Peterson P.A. Fung-Leung W-P. Functional loss of ABCA1 in mice causes severe placental malformation, aberrant lipid distribution, and kidney glomerulonephritis as well as high-density lipoprotein cholesterol deficiency.Am. J. Pathol. 2000; 157: 1017-1029Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar), biliary cholesterol secretion, and fecal sterol output were apparently normal and not reduced (24Drobnik W. Lindenthal B. Lieser B. Ritter M. Christiansen Weber T. Liebisch G. Giesa U. Igel M. Borsukova H. Büchler C. et al.ATP-binding cassette transporter A1 (ABCA1) affects total body sterol metabolism.Gastroenterology. 2001; 120: 1203-1211Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 25Groen A.K. Bloks V.W. Bandsma R.H.J. Ottenhoff R. Chimini G. Kuipers F. Heptobiliary cholesterol transport is not impaired in Abca1-null mice lacking HDL.J. Clin. Invest. 2001; 108: 843-850Crossref PubMed Scopus (143) Google Scholar–26Kosters A. Frijters R.J.J.M. Schaap F.G. Vink E. Plösch T. Ottenhoff R. Jirsa M. De Cuyper I.M. Kuipers F. Groen A.K. Relation between hepatic expression of ATP-binding cassette transporters G5 and G8 and biliary cholesterol secretion in mice.J. Hepatol. 2003; 38: 710-716Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Furthermore, measurements of HMG-CoA reductase activity suggested that sterol synthesis in several peripheral organs was actually increased and not suppressed (24Drobnik W. Lindenthal B. Lieser B. Ritter M. Christiansen Weber T. Liebisch G. Giesa U. Igel M. Borsukova H. Büchler C. et al.ATP-binding cassette transporter A1 (ABCA1) affects total body sterol metabolism.Gastroenterology. 2001; 120: 1203-1211Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). From very limited data, it appeared that patients with Tangier disease also excreted essentially normal amounts of fecal sterols (personal observation by Prof. Gerd Assmann). Thus, these experimental observations suggested that ABCA1 might not play an important role in the massive flux of cholesterol that moves daily out of the peripheral organs. Because of the critical importance of this pathway for understanding overall sterol homeostasis in the whole animal and human, these studies were undertaken to quantitate each step in this pathway for the movement of cholesterol from the peripheral organs to the sites of excretion. Using mice lacking ABCA1 function, the size of the cholesterol pool and the rate of cholesterol export were measured in each organ in vivo. These rates were compared with independent measurements of the amounts of sterol that were delivered to the endocrine glands and liver from HDL and to the amount of cholesterol excreted from the body in the feces as either acidic or neutral sterols. These quantitative measurements establish, as suggested by the earlier publications, that turnover of cholesterol in the peripheral organs and the centripetal flow of this sterol to the sites of excretion are unaffected by whether ABCA1 is functioning or not. The mice used in these experiments were derived from animals kindly provided by Dr. Trudy A. Christiansen-Weber (R.W. Johnson Pharmaceutical Research Institute, San Diego, CA) (23Christiansen-Weber T.A. Voland J.R. Wu Y. Ngo K. Roland B.L. Nguyen S. Peterson P.A. Fung-Leung W-P. Functional loss of ABCA1 in mice causes severe placental malformation, aberrant lipid distribution, and kidney glomerulonephritis as well as high-density lipoprotein cholesterol deficiency.Am. J. Pathol. 2000; 157: 1017-1029Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). These mice were supplied to us as heterozygotes on a C57BL/6:129/Ola background. They were then used to generate abca1−/− mice that were, in turn, bred with C57BL/6:129/Sv wild-type mice. This was done over 10 generations so that, ultimately, the mutation was carried on a predominantly C57BL/6:129/Sv background. The genotypes were identified using PCR analysis as described (23Christiansen-Weber T.A. Voland J.R. Wu Y. Ngo K. Roland B.L. Nguyen S. Peterson P.A. Fung-Leung W-P. Functional loss of ABCA1 in mice causes severe placental malformation, aberrant lipid distribution, and kidney glomerulonephritis as well as high-density lipoprotein cholesterol deficiency.Am. J. Pathol. 2000; 157: 1017-1029Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). All animals were housed in plastic colony cages in rooms with alternating periods of light and dark. After weaning and genotyping by the end of the third week, the animals were fed ad libitum a low cholesterol (0.02%, w/w) pelleted diet (No. 7001; Harlan-Teklad, Madison, WI) until they were studied at 3 months of age. All experiments were carried out during the fed state near the end of the dark phase and used female animals. All experimental protocols were approved by the Institutional Animal Care and Use Committee at the University of Texas Southwestern Medical School. Mouse plasma was harvested from both male and female mice lacking LDLR activity (ldlr−/−) that had been maintained on the low-cholesterol basal diet. The LDL and HDL fractions were isolated by preparative ultracentrifugation in the density ranges of 1.020–1.055 and 1.063–1.21 g/ml, respectively. The LDL preparation was then radiolabeled in the protein moiety with either 125I-tyramine cellobiose or with 131I (27Turley S.D. Spady D.K. Dietschy J.M. Role of liver in the synthesis of cholesterol and the clearance of low density lipoproteins in the cynomolgus monkey.J. Lipid Res. 1995; 36: 67-79Abstract Full Text PDF PubMed Google Scholar, 28Osono Y. Woollett L.A. Herz J. Dietschy J.M. Role of the low density lipoprotein receptor in the flux of cholesterol through the plasma and across the tissues of the mouse.J. Clin. Invest. 1995; 95: 1124-1132Crossref PubMed Scopus (163) Google Scholar–29Glass C. Pittman R.C. Weinstein D.B. Steinberg D. 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HDL-C was labeled with either the intracellularly trapped [1α, 2α(n)-3H]cholesteryl oleyl ether or [cholesteryl-4-14C]oleate by exchange from donor liposomes as described (31Hough J.L. Zilversmit D.B. Comparison of various methods for in vitro cholesteryl ester labeling of lipoproteins from hypercholesterolemic rabbits.Biochim. Biophys. Acta. 1984; 792: 338-347Crossref PubMed Scopus (32) Google Scholar, 32Pittman R.C. Knecht T.P. Rosenbaum M.S. Taylor Jr., C.A. A nonendocytotic mechanism for the selective uptake of high density lipoprotein-associated cholesterol esters.J. Biol. Chem. 1987; 262: 2443-2450Abstract Full Text PDF PubMed Google Scholar–33Woollett L.A. Spady D.K. Kinetic parameters for high density lipoprotein apoprotein AI and cholesteryl ester transport in the hamster.J. Clin. Invest. 1997; 99: 1704-1713Crossref PubMed Scopus (45) Google Scholar). Freshly collected plasma from cholesteryl ester transfer protein transgenic mice was used as a source of CETP. The labeled HDL was reisolated by ultracentrifugation (d = 1.07–1.21 g/ml) and dialyzed against saline. Both labeled LDL and HDL preparations were used within 48 h of preparation. Mice were anesthetized, and a catheter was inserted into the jugular vein. After awakening, each animal was given a bolus of 125I-tyramine cellobiose-labeled LDL followed by a continuous infusion of the same preparation at a rate calculated to maintain a constant specific activity in the plasma (34Spady D.K. Bilheimer D.W. Dietschy J.M. Rates of receptor-dependent and -independent low density lipoprotein uptake in the hamster.Proc. Natl. Acad. Sci. USA. 1983; 80: 3499-3503Crossref PubMed Scopus (162) Google Scholar). Ten minutes before termination of the 4 h infusion period, a bolus of 131I-labeled LDL was administered to each animal. The mice were exsanguinated at the end of the 4 h infusions, and all tissues were removed. The remaining carcass was cut into small pieces. Tissue and plasma samples were then assayed for their content of 125I and 131I (28Osono Y. Woollett L.A. Herz J. Dietschy J.M. Role of the low density lipoprotein receptor in the flux of cholesterol through the plasma and across the tissues of the mouse.J. Clin. Invest. 1995; 95: 1124-1132Crossref PubMed Scopus (163) Google Scholar, 35Xie C. Turley S.D. Dietschy J.M. Cholesterol accumulation in tissues of the Niemann-Pick type C mouse is determined by the rate of lipoprotein-cholesterol uptake through the coated-pit pathway in each organ.Proc. Natl. Acad. Sci. USA. 1999; 96: 11992-11997Crossref PubMed Scopus (85) Google Scholar). A similar procedure was used for HDL clearance measurements. These animals were administered a priming dose of [3H]cholesteryl oleyl ether-labeled HDL followed by a continuous infusion of the same radiolabeled lipoprotein for 4 h. Animals were exsanguinated 10 min after intravenously injecting [14C]cholesteryl oleate-labeled HDL. Plasma, tissue samples, and the remaining carcass were saponified in alcoholic KOH, and sterols were extracted and assayed for their 3H and 14C content. The rates of clearance of LDL and HDL by various tissues were expressed as the microliter of plasma cleared of its LDL or HDL content per hour per gram wet weight (μl/h/g) (34Spady D.K. Bilheimer D.W. Dietschy J.M. Rates of receptor-dependent and -independent low density lipoprotein uptake in the hamster.Proc. Natl. Acad. Sci. USA. 1983; 80: 3499-3503Crossref PubMed Scopus (162) Google Scholar). Using the steady-state plasma concentrations of cholesterol in the LDL and HDL fractions, these clearance values were also used to calculate the amount of total cholesterol and cholesteryl ester, respectively, taken up into each organ from LDL and HDL. These values were expressed as the milligram of sterol taken up per day per kilogram of body weight (mg/day/kg). Each animal was injected intraperitoneally with approximately 40 mCi of [3H]water. One hour later, the animals were anesthetized and exsanguinated. The tissues and remaining carcass were saponified, and the digitonin-precipitable sterols were isolated as described (27Turley S.D. Spady D.K. Dietschy J.M. Role of liver in the synthesis of cholesterol and the clearance of low density lipoproteins in the cynomolgus monkey.J. Lipid Res. 1995; 36: 67-79Abstract Full Text PDF PubMed Google Scholar). The rates of sterol synthesis in each of these tissues were then expressed as the nmol of [3H]water incorporated into digitonin-precipitable sterols each hour per gram wet weight of tissue (nmol/h/g). These rates of incorporation of [3H]water into sterols by tissues were also converted to an equivalent number of milligrams of cholesterol synthesized, assuming 0.69 3H atoms were incorporated into the sterol molecule for each carbon atom (36Turley S.D. Andersen J.M. Dietschy J.M. Rates of sterol synthesis and uptake in the major organs of the rat in vivo.J. Lipid Res. 1981; 22: 551-569Abstract Full Text PDF PubMed Google Scholar, 37Dietschy J.M. Spady D.K. Measurement of rates of cholesterol synthesis using tritiated water.J. Lipid Res. 1984; 25: 1469-1476Abstract Full Text PDF PubMed Google Scholar). These rates were expressed as the milligrams of cholesterol synthesized per day per kilogram of body weight (mg/day/kg). Fractional (%) cholesterol absorption was measured by a fecal dual-isotope ratio method using [4-14C]cholesterol (Perkin-Elmer Life Sciences) and [5,6-3H]sitostanol (American Radiolabeled Chemicals, St. Louis, MO) as described (38Schwarz M. Russell D.W. Dietschy J.M. Turley S.D. Marked reduction in bile acid synthesis in cholesterol 7α-hydroxylase-deficient mice does not lead to diminished tissue cholesterol turnover or to hypercholesterolemia.J. Lipid Res. 1998; 39: 1833-1843Abstract Full Text Full Text PDF PubMed Google Scholar). Bile acid pool size was determined using a HPLC method as the total bile acid content of the small intestine, gall bladder, and liver combined (38Schwarz M. Russell D.W. Dietschy J.M. Turley S.D. Marked reduction in bile acid synthesis in cholesterol 7α-hydroxylase-deficient mice does not lead to diminished tissue cholesterol turnover or to hypercholesterolemia.J. Lipid Res. 1998; 39: 1833-1843Abstract Full Text Full Text PDF PubMed Google Scholar). Pool size was expressed as milligram per kilogram of body weight (mg/kg). Stools were collected from individual animals over a 72 h period. These were dried, weighed, and ground. The rates of fecal bile acid and neutral sterol excretion were measured by an enzymatic method and gas-liquid chromatography, respectively (38Schwarz M. Russell D.W. Dietschy J.M. Turley S.D. Marked reduction in bile acid synthesis in cholesterol 7α-hydroxylase-deficient mice does not lead to diminished tissue cholesterol turnover or to hypercholesterolemia.J. Lipid Res. 1998; 39: 1833-1843Abstract Full Text Full Text PDF PubMed Google Scholar), and are expressed as milligrams of sterol excreted each day per kilogram of body weight (mg/day/kg). The total sterol excretion was calculated as the sum of the acidic and neutral sterol outputs for each animal. The total plasma cholesterol concentration was measured enzymatically (Kit No. 1127771; Boehringer Mannheim, Indianapolis, IN). Plasma triacylglycerol concentrations were measured using Infinity Triglycerides Liquid Stable reagent (ThermoTrace, Noble Park, Australia) (39Repa J.J. Turley S.D. Quan G. Dietschy J.M. Delineation of molecular changes in intrahepatic cholesterol metabolism resulting from diminished cholesterol absorption.J. Lipid Res. 2005; 46: 779-789Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Plasma lipoproteins were separated by fast-protein liquid chromatography using a Sepharose 6 column (33Woollett L.A. Spady D.K. Kinetic parameters for high density lipoprotein apoprotein AI and cholesteryl ester transport in the hamster.J. Clin. Invest. 1997; 99: 1704-1713Crossref PubMed Scopus (45) Google Scholar, 35Xie C. Turley S.D. Dietschy J.M. Cholesterol accumulation in tissues of the Niemann-Pick type C mouse is determined by the rate of lipoprotein-cholesterol uptake through the coated-pit pathway in each organ.Proc. Natl. Acad. Sci. USA. 1999; 96: 11992-11997Crossref PubMed Scopus (85) Google Scholar). The cholesterol content in each fraction was quantitated enzymatically. The organs and remaining carcass were saponified, and their sterols were extracted with petroleum ether. The total cholesterol concentration was determined by gas-liquid chromatography (40Turley S.D. Herndon M.W. Dietschy J.M. Reevaluation and application of the dual-isotope plasma ratio method for the measurement of intestinal cholesterol absorption in the hamster.J. Lipid Res. 1994; 35: 328-339Abstract Full Text PDF PubMed Google Scholar). Plasma corticosterone, estradiol, progesterone, and testosterone concentrations were determined by a commercial facility (Endocrine Services Laboratory, Oregon Regional Primate Research Center, Beaverton, OR). While the various rate constants for cholesterol synthesis and uptake were all usually expressed per hour per gram wet weight of tissue, these same data were also expressed per day per organ and were then normalized to a constant body weight of 1 kg. Thus, all values are also shown in various figures as milligrams of cholesterol synthesized or taken up by each organ each day per kilogram of body weight (mg/day/kg). The data in all of these experiments are presented as the mean ± 1 SEM. The unpaired Student's t-test was used to compare various sets of data, and an asterisk indicates a value that is significantly different (P < 0.05) from its corresponding control value. These studies were designed to measure both the absolute rates of cholesterol flux out of the individual organs into the blood each day as well as the absolute rates of fecal sterol excretion from the whole animal. As cholesterol turnover in organs like the liver and intestine is very different from turnover in more distal organs like the kidney and lung, the various tissues of the body were classified into two, presumably functionally different, groups of organs. Tissues like muscle, lung, kidney, and central nervous system, that presumably depend upon the movement of cholesterol from the cells of these organs to HDL for delivery to the liver, are referred to as peripheral tissues. Organs like liver, small intestine, and other parts of the gastrointestinal tract that can excrete sterol directly into the intestinal lumen, are referred to as central tissues. The most relevant measurement, therefore, for these studies was to quantitate the absolute rates of cholesterol flux from the individual peripheral tissues to the plasma in the presence and absence of ABCA1 function. Preliminary studies revealed similar abnormalities in lipid metabolism in both male and female abca1−/− mice. However, because of the quantitative nature of these studies, all measurements of cholesterol flux were carried out in female animals of the same age. At 3 months of age, these abca1−/− mice showed the expected abnormalities in circulating lipid levels (22McNeish
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