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• W2155518349 abstract "Although the pool of cholesterol in the adult central nervous system (CNS) is large and of constant size, little is known of the process(es) involved in regulation of sterol turnover in this pool. In 7-week-old mice, net excretion of cholesterol from the brain equaled 1.4 mg/day/kg body weight, and from the whole animal was 179 mg/day/kg. Deletion of cholesterol 24-hydroxylase, an enzyme highly expressed in the CNS, did not alter brain growth or myelination, but reduced sterol excretion from the CNS 64% to 0.5 mg/day/kg. In mice with a mutation in the Niemann-Pick C gene that had ongoing neurodegeneration, sterol excretion from the CNS was increased to 2.3 mg/day/kg. Deletion of cholesterol 24-hydroxylase activity in these animals reduced net excretion only 22% to 1.8 mg/day/kg.Thus, at least two different pathways promote net sterol excretion from the CNS. One uses cholesterol 24-hydroxylase and may reflect sterol turnover in large neurons in the brain. The other probably involves the movement of cholesterol or one of its metabolites across the blood-brain barrier and may more closely mirror sterol turnover in pools such as glial cell membranes and myelin. Although the pool of cholesterol in the adult central nervous system (CNS) is large and of constant size, little is known of the process(es) involved in regulation of sterol turnover in this pool. In 7-week-old mice, net excretion of cholesterol from the brain equaled 1.4 mg/day/kg body weight, and from the whole animal was 179 mg/day/kg. Deletion of cholesterol 24-hydroxylase, an enzyme highly expressed in the CNS, did not alter brain growth or myelination, but reduced sterol excretion from the CNS 64% to 0.5 mg/day/kg. In mice with a mutation in the Niemann-Pick C gene that had ongoing neurodegeneration, sterol excretion from the CNS was increased to 2.3 mg/day/kg. Deletion of cholesterol 24-hydroxylase activity in these animals reduced net excretion only 22% to 1.8 mg/day/kg. Thus, at least two different pathways promote net sterol excretion from the CNS. One uses cholesterol 24-hydroxylase and may reflect sterol turnover in large neurons in the brain. The other probably involves the movement of cholesterol or one of its metabolites across the blood-brain barrier and may more closely mirror sterol turnover in pools such as glial cell membranes and myelin. The cells of all extrahepatic tissues outside of the central nervous system (CNS) continuously acquire cholesterol from two sources, de novo synthesis and the uptake of sterol carried in LDLs. In the mouse, as in other species (1Spady D.K. Dietschy J.M. Sterol synthesis in vivo in 18 tissues of the squirrel monkey, guinea pig, rabbit, hamster and rat.J. Lipid Res. 1983; 24: 303-315Abstract Full Text PDF PubMed Google Scholar), the predominant source for this sterol is de novo synthesis (∼100 mg/day/kg body weight), while cellular uptake of LDL cholesterol through the clathrin-coated pit/lysosomal pathway contributes only a small amount (∼5–10 mg/day/kg) to this acquisition process (2Osono 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 (164) Google Scholar, 3Xie 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 (86) Google Scholar, 4Dietschy J.M. Turley S.D. Control of cholesterol turnover in the mouse.J. Biol. Chem. 2002; 277: 3801-3804Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). Within the cells of these tissues, this newly acquired sterol is transported outwardly to the cell surface, where it apparently continuously replaces cholesterol within the bulk phase and sphingolipid-rich microdomains of the plasma membrane (5Simons K. Ikonen E. How cells handle cholesterol.Science. 2000; 290: 1721-1726Crossref PubMed Scopus (1078) Google Scholar, 6Schütz G.J. Kada G. Pastushenko V.P. Schindler H. Properties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy.EMBO J. 2000; 19: 892-901Crossref PubMed Scopus (487) Google Scholar). Because the pool of cholesterol in these extrahepatic tissues remains constant (∼2,200 mg/kg), each day an amount of sterol equal to that newly acquired must be removed from these cells and carried by lipoproteins back to the liver for excretion from the body as either neutral, i.e., cholesterol or acidic, i.e., bile acid, sterols. While it is not entirely clear why the integrity of these cells depends upon this continuous flow of sterol from the endoplasmic reticulum and lysosomes to the plasma membrane, several lines of evidence suggest this movement may be involved in the regulation of certain proteins and in the function of a number of specific transporters on the cell surface (7Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Caveolins, liquid-ordered domains, and signal transduction.Mol. Cell. Biol. 1999; 19: 7289-7304Crossref PubMed Scopus (926) Google Scholar, 8Fassbender K. Simons M. Bergmann C. Stoick M. Lütjohann D. Keller P. Runz H. Kühl S. Bertsch T. von Bergmann K. Hennerich M. Beyreuther K. Hartmann T. Simvastatin strongly reduces levels of Alzheimer's disease b-amyloid peptides Ab42 and Ab40 in vitro and in vivo.Proc. Natl. Acad. Sci. USA. 2001; 98: 8856-8861Crossref PubMed Scopus (1036) Google Scholar, 9Sowa G. Pypaert M. Sessa W.C. Distinction between signaling mechanisms in lipid rafts vs. caveolae.Proc. Natl. Acad. Sci. USA. 2001; 98: 14072-14077Crossref PubMed Scopus (192) Google Scholar, 10Hao M. Mukherjee S. Maxfield F.R. Cholesterol depletion induces large scale domain segregation in living cell membranes.Proc. Natl. Acad. Sci. USA. 2001; 98: 13072-13077Crossref PubMed Scopus (251) Google Scholar, 11Ros-Baró A. López-Iglesias C. Peiró S. Bellido D. Palacín M. Zorzano A. Camps M. Lipid rafts are required for GLUT4 internalization in adipose cells.Proc. Natl. Acad. Sci. USA. 2001; 98: 12050-12055Crossref PubMed Scopus (125) Google Scholar). Because of this continuous replacement, in a species with a very high metabolic rate like the mouse, ∼7–9% of the total body pool of cholesterol is turned over each day (2Osono 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 (164) Google Scholar, 4Dietschy J.M. Turley S.D. Control of cholesterol turnover in the mouse.J. Biol. Chem. 2002; 277: 3801-3804Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). Much less is understood about cholesterol turnover in the brain. As in most of the other extrahepatic organs, sterol in the brain is essentially all unesterified, but unlike in these other tissues, this cholesterol is present in two functionally distinct pools, the plasma membranes of glial and nerve cells, and the multi-layered myelin sheaths elaborated by support cells that surround the processes of neurons. While the concentration of unesterified cholesterol in most organs varies from ∼1.4 mg/g wet weight (muscle) to ∼5.0 mg/g (lung) (2Osono 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 (164) Google Scholar), in the brain, these concentrations vary from ∼8 mg/g (gray matter) to ∼40 mg/g (spinal cord). In the mouse, the pool of cholesterol in the brain (∼330 mg/kg) accounts for about 15% of the total body pool, but in the primate, over 20% of the body sterol pool is located in the CNS (12Cook R.P. Distribution of sterols in organisms and in tissues.in: Cook R.P. Cholesterol. Chemistry, Biochemistry, and Pathology. Academic Press, Inc., New York1958: 145-180Crossref Google Scholar). Nevertheless, the turnover of this large pool of unesterified cholesterol within the CNS is apparently very slow. In the mouse, for example, where 7–9% of the whole animal cholesterol pool is replaced each day, only about 0.4% of the pool in the brain is turned over (13Dietschy J.M. Turley S.D. Cholesterol metabolism in the brain.Curr. Opin. Lipidol. 2001; 12: 105-112Crossref PubMed Scopus (739) Google Scholar). Similarly slow rates of sterol turnover have been reported in the brains of the baboon and human (13Dietschy J.M. Turley S.D. Cholesterol metabolism in the brain.Curr. Opin. Lipidol. 2001; 12: 105-112Crossref PubMed Scopus (739) Google Scholar, 14Wilson J.D. The measurement of the exchangeable pools of cholesterol in the baboon.J. Clin. Invest. 1970; 49: 655-665Crossref PubMed Scopus (53) Google Scholar, 15Björkhem I. Lütjohann D. Diczfalusy U. Ståhle L. Ahlborg G. Wahren J. Cholesterol homeostasis in human brain: turnover of 24S-hydroxycholesterol and evidence for a cerebral origin of most of this oxysterol in the circulation.J. Lipid Res. 1998; 39: 1594-1600Abstract Full Text Full Text PDF PubMed Google Scholar). The processes responsible for this turnover, however, are poorly understood, and it is not clear whether these overall rates of turnover apply equally to the pools of cholesterol in the plasma membranes and myelin sheaths within the CNS. In contrast to other tissues of the body, acquisition of cholesterol within the brain appears to come entirely from de novo synthesis. With the development of methods that allowed measurement of absolute rates of synthesis in vivo (16Jeske D.J. Dietschy J.M. Regulation of rates of cholesterol synthesis in vivo in the liver and carcass of the rat measured using [3H]water.J. Lipid Res. 1980; 21: 364-376Abstract Full Text PDF PubMed Google Scholar), it was found in several species that the rate of accumulation of sterol in the developing CNS could be fully accounted for by the rate of de novo synthesis (17Edmond J. Korsak R.A. Morrow J.W. Torok-Both G. Catlin D.H. Dietary cholesterol and the origin of cholesterol in the brain of developing rats.J. Nutr. 1991; 121: 1323-1330Crossref PubMed Scopus (91) Google Scholar, 18Jurevics H. Morell P. Cholesterol for synthesis of myelin is made locally, not imported into brain.J. Neurochem. 1995; 64: 895-901Crossref PubMed Scopus (251) Google Scholar, 19Jurevics H.A. Kidwai F.Z. Morell P. Sources of cholesterol during development of the rat fetus and fetal organs.J. Lipid Res. 1997; 38: 723-733Abstract Full Text PDF PubMed Google Scholar, 20Turley S.D. Burns D.K. Dietschy J.M. Preferential utilization of newly synthesized cholesterol for brain growth in neonatal lambs.Am. J. Physiol. 1998; 274: E1099-E1105Crossref PubMed Google Scholar). Furthermore, there was a very close correlation between the rate of synthesis and the ultimate concentration of sterol found in different regions of the CNS (20Turley S.D. Burns D.K. Dietschy J.M. Preferential utilization of newly synthesized cholesterol for brain growth in neonatal lambs.Am. J. Physiol. 1998; 274: E1099-E1105Crossref PubMed Google Scholar, 21Turley S.D. Dietschy J.M. Regional variation in cholesterol synthesis and low density lipoprotein transport in the brain of the fetus, newborn and adult animal.Nutr. Metab. Cardiovasc. Dis. 1997; 7: 195-201Google Scholar). In contrast, repeated attempts to demonstrate LDL cholesterol (or HDL cholesterol) uptake into the brain, even in the fetus before closing of the blood-brain barrier, failed to show net transfer of sterol from the plasma into the CNS (2Osono 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 (164) Google Scholar, 21Turley S.D. Dietschy J.M. Regional variation in cholesterol synthesis and low density lipoprotein transport in the brain of the fetus, newborn and adult animal.Nutr. Metab. Cardiovasc. Dis. 1997; 7: 195-201Google Scholar, 22Turley S.D. Burns D.K. Rosenfeld C.R. Dietschy J.M. Brain does not utilize low density lipoprotein-cholesterol during fetal and neonatal development in the sheep.J. Lipid Res. 1996; 37: 1953-1961Abstract Full Text PDF PubMed Google Scholar). Thus, the tissues of the CNS have very high rates of cholesterol synthesis during brain development in the fetus and newborn, and these rates decline with maturation of the animal (23Dietschy 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). However, cholesterol synthesis continues, albeit at a low rate, even after mature brain size is achieved and the pools of sterol in the CNS become constant (1Spady D.K. Dietschy J.M. Sterol synthesis in vivo in 18 tissues of the squirrel monkey, guinea pig, rabbit, hamster and rat.J. Lipid Res. 1983; 24: 303-315Abstract Full Text PDF PubMed Google Scholar, 23Dietschy 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). Several lines of evidence suggest that this newly synthesized cholesterol is involved in an internal circulation of sterol among different cell types within the brain. The glia and neurons of the CNS express several members of the LDL receptor (LDLR) family, including the LDLR itself and the LDLR related protein, and the cerebrospinal fluid contains a number of apolipoproteins including apolipoprotein E (apoE), apoA-I, and apoA-IV (24Pitas R.E. Boyles J.K. Lee S.H. Hui D. Weisgraber K.H. Lipoproteins and their receptors in the central nervous system. Characterization of the lipoproteins in cerebrospinal fluid and identification of apolipoprotein B, E(LDL) receptors in the brain.J. Biol. Chem. 1987; 262: 14352-14360Abstract Full Text PDF PubMed Google Scholar, 25Borghini I. Barja F. Pometta D. James R.W. Characterization of subpopulations of lipoprotein particles isolated from human cerebrospinal fluid.Biochim. Biophys. Acta. 1995; 1255: 192-200Crossref PubMed Scopus (150) Google Scholar, 26Herz J. Bock H.H. Lipoprotein receptors in the nervous system.Annu. Rev. Biochem. 2002; 71: 405-434Crossref PubMed Scopus (358) Google Scholar). In one study, for example, it was shown that synapse formation between neurons in vitro required the presence of glial cells; however, this requirement could be obviated by providing the nerve cells with cholesterol complexed to apoE (27Mauch D.H. Nägler K. Schumacher S. Göritz C. Müller E-C. Otto A. Pfrieger F.W. CNS synaptogenesis promoted by glia-derived cholesterol.Science. 2001; 294: 1354-1357Crossref PubMed Scopus (1271) Google Scholar). That neurons take up such apoE-cholesterol complexes is also consistent with the observation that unesterified cholesterol accumulates in these cells when Niemann-Pick type C (NPC1), a protein required for the processing of sterol entering cells through the clathrin-coated pit pathway, is mutated (28Xie C. Turley S.D. Pentchev P.G. Dietschy J.M. Cholesterol balance and metabolism in mice with loss of function of Niemann-Pick C protein.Am. J. Physiol. 1999; 276: E336-E344PubMed Google Scholar, 29Xie C. Burns D.K. Turley S.D. Dietschy J.M. Cholesterol is sequestered in the brains of mice with Niemann-Pick Type C disease but turnover is increased.J. Neuropathol. Exp. Neurol. 2000; 59: 1106-1117Crossref PubMed Scopus (98) Google Scholar). If, as these observations suggest, sterol is being continuously synthesized within the CNS even in the mature animal where the pool of sterol in the brain has become constant, it follows that mechanisms must be available to continuously transport cholesterol across the blood-brain barrier into the plasma for excretion. In principle, such movement might involve the transfer of cholesterol itself or the movement of this sterol after hydroxylation within the CNS. The observations that the brain contains both cholesterol 24-hydroxylase and sterol 27-hydroxylase activities (30Pedersen J.I. Oftebro H. Björkhem I. Reconstitution of C27-steroid 26-hydroxylase activity from bovine brain mitochondria.Biochem. Int. 1989; 18: 615-622PubMed Google Scholar, 31Björkhem I. Lütjohann D. Breuer O. Sakinis A. Wennmalm Å. Importance of a novel oxidative mechanism for elimination of brain cholesterol.J. Biol. Chem. 1997; 272: 30178-30184Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 32Zhang J. Akwa Y. El-Etr M. Baulieu E-E. Sjövall J. Metabolism of 27-, 25- and 24-hydroxycholesterol in rat glial cells and neurons.Biochem. J. 1997; 322: 175-184Crossref PubMed Scopus (68) Google Scholar), and that there is net transfer of 24(S)-hydroxycholesterol from the brain into the venous outflow in humans (33Lütjohann D. Breuer O. Ahlborg G. Nennesmo I. Sidén Å. Diczfalusy U. Björkhem I. Cholesterol homeostasis in human brain: evidence for an age-dependent flux of 24S-hydroxycholesterol from the brain into the circulation.Proc. Natl. Acad. Sci. USA. 1996; 93: 9799-9804Crossref PubMed Scopus (573) Google Scholar) indicate that formation of these oxysterols may be one of the major pathways for the movement of sterol out of the CNS. With the availability of mice lacking cholesterol 24-hydroxylase activity (Cyp46a1−/−) (34Lund E.G. Guileyardo J.M. Russell D.W. cDNA cloning of cholesterol 24-hydroxylase, a mediator of cholesterol homeostasis in the brain.Proc. Natl. Acad. Sci. USA. 1999; 96: 7238-7243Crossref PubMed Scopus (534) Google Scholar, 35Lund E.G. Xie C. Kotti T. Turley S.D. Dietschy J.M. Russell D.W. Knockout of the cholesterol 24-hydroxylase gene in mice reveals a brain-specific mechanism of cholesterol turnover.J. Biol. Chem. 2003; 278: 22980-22988Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar), as well as those lacking cholesterol 7α-hydroxylase (Cyp7a1−/−) and sterol 27-hydroxylase (Cyp27a1−/−) activities (36Schwarz M. Russell D.W. Dietschy J.M. Turley S.D. Marked reduction in bile acid synthesis in cholesterol 7a-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, 37Repa J.J. Lund E.G. Horton J.D. Leitersdorf E. Russell D.W. Dietschy J.M. Turley S.D. Disruption of the sterol 27-hydroxylase gene in mice results in hepatomegaly and hypertriglyceridemia. Reversal by cholic acid feeding.J. Biol. Chem. 2000; 275: 39685-39692Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar), it is now possible to measure the quantitative importance of each of these pathways for cholesterol turnover in the brain. The current studies, therefore, were undertaken 1) to explore the effect of age and gender on cholesterol metabolism in the mouse brain; 2) to measure the effect of deletion of CYP46A1 and the other sterol hydroxylases on cholesterol metabolism in different regions of the CNS; 3) to quantitate net sterol turnover in the CNS and to measure the role of CYP46A1 in this process; and, finally, 4) to measure the rate of net cholesterol turnover in animals with ongoing neurodegeneration and to define the role of CYP46A1 in this excretory process. The mice with deletion of either cholesterol 24-hydroxylase (Cyp46a1−/−) or cholesterol 7α-hydroxylase (Cyp7a1−/−) activity were generated as described previously (35Lund E.G. Xie C. Kotti T. Turley S.D. Dietschy J.M. Russell D.W. Knockout of the cholesterol 24-hydroxylase gene in mice reveals a brain-specific mechanism of cholesterol turnover.J. Biol. Chem. 2003; 278: 22980-22988Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 36Schwarz M. Russell D.W. Dietschy J.M. Turley S.D. Marked reduction in bile acid synthesis in cholesterol 7a-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, 38Ishibashi S. Schwarz M. Frykman P.K. Herz J. Russell D.W. Disruption of cholesterol 7a-hydroxylase gene in mice: I. Postnatal lethality reversed by bile acid and vitamin supplementation.J. Biol. Chem. 1996; 271: 18017-18023Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). The sterol 27-hydroxylase knockout mice (Cyp27a1−/−) were originally obtained from Dr. Eran Leitersdorf (37Repa J.J. Lund E.G. Horton J.D. Leitersdorf E. Russell D.W. Dietschy J.M. Turley S.D. Disruption of the sterol 27-hydroxylase gene in mice results in hepatomegaly and hypertriglyceridemia. Reversal by cholic acid feeding.J. Biol. Chem. 2000; 275: 39685-39692Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 39Rosen H. Reshef A. Maeda N. Lippoldt A. Shpizen S. Triger L. Eggertsen G. Björkhem I. Leitersdorf E. Markedly reduced bile acid synthesis but maintained levels of cholesterol and vitamin D metabolites in mice with disrupted sterol 27-hydroxylase gene.J. Biol. Chem. 1998; 273: 14805-14812Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Mice with a mutation in NPC1 protein (Npc1−/−) were obtained from a colony at the National Institutes of Health (28Xie C. Turley S.D. Pentchev P.G. Dietschy J.M. Cholesterol balance and metabolism in mice with loss of function of Niemann-Pick C protein.Am. J. Physiol. 1999; 276: E336-E344PubMed Google Scholar, 40Loftus S.K. Morris J.A. Carstea E.D. Gu J.Z. Cummings C. Brown A. Ellison J. Ohno K. Rosenfeld M.A. Tagle D.A. Pentchev P.G. Pavan W.J. Murine model of Niemann-Pick C disease: mutation in a cholesterol homeostasis gene.Science. 1997; 277: 232-235Crossref PubMed Scopus (705) Google Scholar) and were then bred into a mixed background of C57BL/6 and 129S6/SvEv. All genetically modified animals were maintained in the same mixed background of C57BL/6 and 129S6/SvEv, as were matching control mice (Cyp46a1+/+, Cyp7a1+/+, Cyp27a1+/+, Npc1+/+). The animals were fed ad libitum a low-cholesterol (0.02%, w/w), low-fat (4%, w/w) basal rodent diet (No. 7001, Harlan Teklad, Madison, WI) after weaning at the end of the third week. With the exception of one experiment in which animals were studied at ages varying from 7 to 210 days, all studies were carried out when the mice were exactly 7 weeks of age and in the fed state near the end of the 12 h dark phase of the light cycle (41Xie C. Turley S.D. Dietschy J.M. Centripetal cholesterol flow from the extrahepatic organs through the liver is normal in mice with mutated Niemann-Pick Type C protein (NPC1).J. Lipid Res. 2000; 41: 1278-1289Abstract Full Text Full Text PDF PubMed Google Scholar). The experimental groups contained nearly equal numbers of males and females because there were no significant gender differences observed in the CNS in any of these measurements. At the termination of these experiments, the entire CNS was removed, including the spinal cord. In some studies, the CNS was divided into five regions identified as cerebrum, cerebellum, mid-brain, brain stem, and spinal cord. All experimental protocols were approved by the Institutional Animal Care and Research Advisory Committee. Animals were exsanguinated from the inferior vena cava, and the CNS was removed and saponified. The cholesterol was extracted and measured by gas-liquid chromotography using stigmastanol as an internal standard (36Schwarz M. Russell D.W. Dietschy J.M. Turley S.D. Marked reduction in bile acid synthesis in cholesterol 7a-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, 42Turley 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). The values were either expressed as mg of cholesterol/g wet weight of tissue (mg/g) or converted to mg of cholesterol in a specific tissue normalized to 1 kg body weight (mg/kg). The mean rate of expansion of the pool of cholesterol in the CNS at 7 weeks of age was determined by measuring the size of this pool per kg body weight in groups of mice exactly 6 and 8 weeks of age. These data were then entered into a linear regression program in order to calculate the average daily accretion rate at 7 weeks expressed as mg of cholesterol accumulated/day/kg (mg/day/kg). Each mouse was injected with 50 mCi of 3H2O intraperitoneally. One hour later, the animals were anesthetized and exsanguinated. The tissues were removed and saponified, and digitonin-precipitable sterols were isolated as described previously. The rates of cholesterol synthesis in each tissue were determined and expressed as the nmol of 3H2O incorporated into sterols/h/g of tissue (nmol/h/g) (2Osono 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 (164) Google Scholar, 36Schwarz M. Russell D.W. Dietschy J.M. Turley S.D. Marked reduction in bile acid synthesis in cholesterol 7a-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). Because the 3H/C incorporation ratio was known, these rates could be converted to absolute rates of cholesterol synthesis and could also be expressed as milligrams of cholesterol synthesized in a particular organ or whole animal/day/kg body weight (mg/day/kg) (16Jeske D.J. Dietschy J.M. Regulation of rates of cholesterol synthesis in vivo in the liver and carcass of the rat measured using [3H]water.J. Lipid Res. 1980; 21: 364-376Abstract Full Text PDF PubMed Google Scholar, 43Dietschy 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). After exsanguination of the mice, aliquots of tissue were immediately removed and frozen in liquid nitrogen. Total RNA was prepared from tissues using the RNA Stat60 reagent (Tel-Test, Friendswood, TX). In one experiment, 5 μg of poly(A)+ RNA was isolated and subjected to electrophoresis, and Northern blotting was performed as described previously (37Repa J.J. Lund E.G. Horton J.D. Leitersdorf E. Russell D.W. Dietschy J.M. Turley S.D. Disruption of the sterol 27-hydroxylase gene in mice results in hepatomegaly and hypertriglyceridemia. Reversal by cholic acid feeding.J. Biol. Chem. 2000; 275: 39685-39692Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). The amount of radioactivity in each signal was analyzed by PhosphorImaging (Molecular Dynamics). In another study, CYP46A1 mRNA was determined by an RNase protection assay using GAPDH as the internal control. Total RNA (40 μg) was hybridized with 32P-labeled riboprobes. Following RNase digestion, the mRNA-protected 32P-labeled probes were separated by electrophoresis, and the radioactivity in each band was quantified using a PhosphorImaging system (44Masliah E. Mallory M. Veinbergs I. Miller A. Samuel W. Alterations in apolipoprotein E expression during aging and neurodegeneration.Prog. Neurobiol. 1996; 50: 493-503Crossref PubMed Scopus (79) Google Scholar). In a third series of experiments, levels of CYP46A1, ATP binding cassette transporter A1 (ABCA1), SR-BI, sterol regulatory element binding protein-1c (SREBP-1c), and SREBP-2 mRNAs were determined by real-time polymerase chain reactions using an Applied Biosystems PRISM 7900HT machine. Immunoblots of the CYP46A1 protein were carried out as described (34Lund E.G. Guileyardo J.M. Russell D.W. cDNA cloning of cholesterol 24-hydroxylase, a mediator of cholesterol homeostasis in the brain.Proc. Natl. Acad. Sci. USA. 1999; 96: 7238-7243Crossref PubMed Scopus (534) Google Scholar). The separated proteins were electroblotted to polyvinylidene difluoride membranes and incubated with T-623 antiserum (1:1,000 to 1:2,000 dilution of unpurified serum). A donkey anti-rabbit horseradish peroxidase-conjugated antibody (Amersham Pharmacia) was used as a secondary antibody. Visualization was via enhanced chemiluminescence kits (Amersham Pharmacia). All data are presented as means ± SEM. The differences between control and genetically modified animals were tested for statistical significance (P < 0.05) by an unpaired, two-tailed Student's t-test. In the figures and table, an asterisk indicates a value that was significantly different at this level from its appropriate control value. Because previous studies in the mouse have shown significant age and gender effects on several aspects of sterol metabolism (41Xie C. Turley S.D. Dietschy J.M. Centripetal cholesterol flow from the extrahepatic organs through the liver is normal in mice with mutated Niemann-Pick Type C protein (NPC1).J. Lipid Res. 2000; 41: 1278-1289Abstract Full Text Full Text PDF PubMed Google Scholar, 45Schwarz M. Russell D.W. Dietschy J.M. Turley S.D. Alternate pathways of bile acid synthesis in the cholesterol 7a-hydroxylase knockout mouse are not upregulated by either cholesterol or cholestyramine feeding.J. Lipid Res. 2001; 42: 1594-1603Abstract Full Text Full Text PDF PubMed Google Scholar), initial measurements were made in the brains of male and female mice of different ages. As" @default.
• W2155518349 created "2016-06-24" @default.
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• W2155518349 date "2003-09-01" @default.
• W2155518349 modified "2023-10-16" @default.
• W2155518349 title "Quantitation of two pathways for cholesterol excretion from the brain in normal mice and mice with neurodegeneration" @default.
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