Control of Cholesterol Turnover in the Mouse

endoplasmic reticulum adenosine triphosphate-binding cassette transporter A1 ABC transporter B4 (MDR2/3) ABC transporter B11 (SPGP) ABC transporter G5 ABC transporter G8 apolipoprotein A-I cholesteryl ester transfer protein cholesterol 7α-hydroxylase sterol 27-hydroxylase high density lipoprotein low density lipoprotein LDL receptor Niemann-Pick type C protein 1 scavenger receptor class B, type I very low density lipoprotein The mouse is now commonly used to explore the physiological changes that take place in sterol metabolism when a specific protein function involved in cholesterol turnover is either deleted or overexpressed. The major pathways for the net flow of cholesterol through the tissue compartments of this species are outlined in Fig. 1. This diagram separates the overall process of cholesterol turnover into those events that occur within the cells of the extrahepatic tissues (90% of body mass), that promote movement through the plasma space (4.6% of body mass), and that are involved in secretion from the liver (5% of body mass) into the intestine. As implied by the thickness of the arrows, most cholesterol is synthesized in the extrahepatic organs and moves from the ER1 to the bulk phase and specialized microdomains of the plasma membrane (1Heino S. Lusa S. Somerharju P. Ehnholm C. Olkkonen V.M. Ikonen E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8375-8380Crossref PubMed Scopus (206) Google Scholar, 2Simons K. Ikonen E. Science. 2000; 290: 1721-1726Crossref PubMed Scopus (1046) Google Scholar, 3Schlegel A. Lisanti M.P. Cytokine Growth Factor Rev. 2001; 12: 41-51Crossref PubMed Scopus (70) Google Scholar). Because cholesterol does not accumulate in these tissues, it must be continuously removed from the plasma membrane and bound to circulating apoA-I, a process recently postulated to be under the control of ABCA1 (4Schmitz G. Langmann T. Curr. Opin. Lipidol. 2001; 12: 129-140Crossref PubMed Scopus (179) Google Scholar, 5Mendez A.J. Lin G. Wade D.P. Lawn R.M. Oram J.F. J. Biol. Chem. 2001; 276: 3158-3166Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Within the plasma space, cholesterol is esterified to CE in the mature HDL particle utilizing the enzyme lecithin:cholesterol acyltransferase. This CE is then selectively removed from the lipoprotein particle by SR-BI primarily in the liver (and endocrine tissues) and hydrolyzed (6Landschulz K.T. Pathak R.K. Rigotti A. Krieger M. Hobbs H.H. J. Clin. Invest. 1996; 98: 984-995Crossref PubMed Scopus (461) Google Scholar, 7Trigatti B.L. Rigotti A. Braun A. Biochim. Biophys. Acta. 2000; 1529: 276-286Crossref PubMed Scopus (55) Google Scholar, 8Xie C. Turley S.D. Dietschy J.M. J. Lipid Res. 2000; 41: 1278-1289Abstract Full Text Full Text PDF PubMed Google Scholar). The resultant cholesterol mixes with other sterols newly synthesized in the liver or absorbed from the diet. A portion of this pool is utilized as substrate for bile acid synthesis or for secretion into the bile and, ultimately, into the intestine. Alternatively, part of this sterol may be esterified by acyl-CoA:cholesterol acyltransferase, incorporated into the nascent VLDL particle, and secreted into the plasma space (9Shelness G.S. Sellers J.A. Curr. Opin. Lipidol. 2001; 12: 151-157Crossref PubMed Scopus (228) Google Scholar). During metabolism of this VLDL particle, a portion of the cholesterol reaches the LDL particle, and a small fraction (∼20%) of this lipoprotein is cleared by the cells of the extrahepatic tissues utilizing the LDLR clustered in clathrin-coated pits (10Osono Y. Woollett L.A. Herz J. Dietschy J.M. J. Clin. Invest. 1995; 95: 1124-1132Crossref PubMed Scopus (163) Google Scholar). After processing through the lysosomal pathway, this small amount of cholesterol joins the much larger pool of newly synthesized cholesterol destined for transport to the plasma membrane (Fig. 1). This scheme emphasizes that most cholesterol is synthesized in the extrahepatic tissues and that the excretion of this sterol from the body largely takes place by secreting cholesterol itself from the liver into the feces or after first metabolizing the cholesterol molecule to bile acid and then secreting this acidic sterol into the feces (11Russell D.W. Biochim. Biophys. Acta. 2000; 1529: 126-135Crossref PubMed Scopus (300) Google Scholar). Significant amounts of cholesterol are also excreted from the body through the sloughing of skin cells and secretions and after conversion to steroid hormones, but these pathways are not shown in Fig. 1. Nevertheless, in the mouse, measuring the output of fecal neutral and acidic sterols always underestimates cholesterol turnover when compared with quantifying sterol input into the animal from dietary cholesterol intake and de novo synthesis. In many respects, cholesterol turnover in the mouse is both quantitatively and qualitatively different from other animal models, particularly primates. As shown in TableI, on a typical, cereal-based animal diet the mouse ingests about 30 mg of cholesterol/day/kg of body weight whereas other species, including humans, usually eat much less (column A). In different strains of mice, as well as in different humans, the amount of this dietary sterol load that is absorbed varies between 30 and 70%. Typically, the mouse synthesizes ∼160 mg/day/kg of cholesterol whereas the human makes only ∼10 mg/day/kg (column B). As in other rodents, the liver of the mouse is relatively more important as a site for this synthesis (∼40%) than is true in the primate (10–12%) (column C). Because the pool of cholesterol in the whole animal is similar in the rodent and human (∼2200 mg/kg) and does not change with age, the total input of sterol from the diet and synthesis in the mouse (∼190 mg/day/kg) is much greater than in the other species and, in particular, is 13-fold higher than in the human (∼15 mg/day/kg). There is a similar, marked difference in the handling of cholesterol carried in circulating LDL. Although the liver is the primary site for the removal of LDL from the plasma in all species (column E), the rate of entry of cholesterol into the LDL pool in the mouse (∼50 mg/day/kg) is only 4-fold higher than in the human (∼13 mg/day/kg) (column D), but the rate of hepatic LDL clearance in this animal (∼500 ml/day/kg) is 40-fold greater than in the human (∼12 ml/day/kg) (12Dietschy J.M. Turley S.D. Spady D.K. J. Lipid Res. 1993; 34: 1637-1659Abstract Full Text PDF PubMed Google Scholar, 13Xie C. Turley S.D. Dietschy J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11992-11997Crossref PubMed Scopus (83) Google Scholar). As a consequence, the steady-state concentration of cholesterol carried in LDL in the mouse is usually ∼7 mg/dl whereas in the human this value usually exceeds 100 mg/dl (column F). Despite these variations, however, most animals, including the mouse, ultimately excrete cholesterol in the feces in approximately equal amounts as neutral (column G) and acidic (column H) sterols.Table IRepresentative values for cholesterol turnover in the mouse and in other speciesA. Dietary cholesterol intakeB. Whole animal cholesterol synthesis rateC. Liver contribution to whole animal synthesisD. Whole animal LDL-cholesterol production rateE. Liver contribution to LDL-cholesterol clearanceF. Steady-state LDL-cholesterol concentrationG. Fecal neutral sterol excretion rateH. Fecal acidic sterol excretion ratemg/day/kgmg/day/kg% totalmg/day/kg% totalmg/dlmg/day/kgmg/day/kg1. Mouse (0.025 kg)30160405080760502. Hamster (0.120 kg)9403528752518243. Monkey (6 kg)51212178055NANA4. Human (70 kg)51010 (?)1-aThe figure in the human giving the percentage contribution of the liver to whole animal cholesterol synthesis was estimated from tissue obtained by biopsy and used to measure rates of synthesis in vitro.137012087This table shows typical values for eight different parameters of cholesterol metabolism measured in vivo in the mouse and compares them with similar values in three other species including the hamster, cynomolgus monkey, and human. All of these values were obtained in animals or humans on a “low” dietary sterol intake where the amount of cholesterol ingested each day was <50% of the rate of de novo cholesterol synthesis in the whole animal. These values were calculated from references (8Xie C. Turley S.D. Dietschy J.M. J. Lipid Res. 2000; 41: 1278-1289Abstract Full Text Full Text PDF PubMed Google Scholar, 12Dietschy J.M. Turley S.D. Spady D.K. J. Lipid Res. 1993; 34: 1637-1659Abstract Full Text PDF PubMed Google Scholar, 13Xie C. Turley S.D. Dietschy J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11992-11997Crossref PubMed Scopus (83) Google Scholar, 27Turley S.D. Schwarz M. Spady D.K. Dietschy J.M. Hepatology. 1998; 28: 1088-1094Crossref PubMed Scopus (81) Google Scholar,33Von Bergmann K. Mok H.Y. Hardison W.G.M. Grundy S.M. Gastroenterology. 1979; 77: 1183-1192Abstract Full Text PDF PubMed Scopus (35) Google Scholar, 34Turley S.D. Spady D.K. Dietschy J.M. J. Lipid Res. 1995; 36: 67-79Abstract Full Text PDF PubMed Google Scholar, 35Turley S.D. Spady D.K. Dietschy J.M. Hepatology. 1997; 25: 797-803Crossref PubMed Scopus (33) Google Scholar, 36Turley S.D. Spady D.K. Dietschy J.M. J. Lipid Res. 1997; 38: 1598-1611Abstract Full Text PDF PubMed Google Scholar) and from unpublished measurements in this laboratory. Values for fecal sterol output in the cynomolgus monkey were not available (NA).1-a The figure in the human giving the percentage contribution of the liver to whole animal cholesterol synthesis was estimated from tissue obtained by biopsy and used to measure rates of synthesis in vitro. Open table in a new tab This table shows typical values for eight different parameters of cholesterol metabolism measured in vivo in the mouse and compares them with similar values in three other species including the hamster, cynomolgus monkey, and human. All of these values were obtained in animals or humans on a “low” dietary sterol intake where the amount of cholesterol ingested each day was <50% of the rate of de novo cholesterol synthesis in the whole animal. These values were calculated from references (8Xie C. Turley S.D. Dietschy J.M. J. Lipid Res. 2000; 41: 1278-1289Abstract Full Text Full Text PDF PubMed Google Scholar, 12Dietschy J.M. Turley S.D. Spady D.K. J. Lipid Res. 1993; 34: 1637-1659Abstract Full Text PDF PubMed Google Scholar, 13Xie C. Turley S.D. Dietschy J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11992-11997Crossref PubMed Scopus (83) Google Scholar, 27Turley S.D. Schwarz M. Spady D.K. Dietschy J.M. Hepatology. 1998; 28: 1088-1094Crossref PubMed Scopus (81) Google Scholar,33Von Bergmann K. Mok H.Y. Hardison W.G.M. Grundy S.M. Gastroenterology. 1979; 77: 1183-1192Abstract Full Text PDF PubMed Scopus (35) Google Scholar, 34Turley S.D. Spady D.K. Dietschy J.M. J. Lipid Res. 1995; 36: 67-79Abstract Full Text PDF PubMed Google Scholar, 35Turley S.D. Spady D.K. Dietschy J.M. Hepatology. 1997; 25: 797-803Crossref PubMed Scopus (33) Google Scholar, 36Turley S.D. Spady D.K. Dietschy J.M. J. Lipid Res. 1997; 38: 1598-1611Abstract Full Text PDF PubMed Google Scholar) and from unpublished measurements in this laboratory. Values for fecal sterol output in the cynomolgus monkey were not available (NA). Fig. 2 summarizes five aspects of cholesterol balance in mice in which the function of a specific protein either has been deleted or overexpressed. For comparison, column A gives values for these different processes in control mice of a mixed genetic background. The pool of cholesterol in such whole animals averages 2175 mg/kg (A1), whereas that portion of this pool circulating in the plasma space is much smaller and averages only 45 mg/kg (at a concentration of 95 mg/dl) (A3). The net centripetal flux of cholesterol from the extrahepatic organs through the plasma space to the liver equals about 92 mg/day/kg (A2) where it mixes with additional amounts of cholesterol derived from hepatic synthesis and the diet. Ultimately, 60 mg/day/kg of cholesterol is excreted as fecal neutral sterols (A4), and 50 mg/day/kg is lost from the body as fecal acidic sterols (A5). Columns B and C, respectively, show the values of these parameters in two animal models with altered activity of proteins that are thought to function in the movement of sterol through the cell and across the plasma membrane. NPC1 is expressed in virtually every tissue and is involved in the movement of cholesterol, derived from the uptake of apoB-containing lipoproteins, from the lysosomal compartment to the pool of newly synthesized sterol destined for insertion into the plasma membrane (Fig. 1) (14Loftus 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. Science. 1997; 277: 232-235Crossref PubMed Scopus (688) Google Scholar, 15Zhang M. Dwyer N.K. Neufeld E.B. Love D.C. Cooney A. Comly M. Patel S. Watari H. Strauss III, J.F. Pentchev P.G. Hanover J.A. Blanchette-Mackie E.J. J. Biol. Chem. 2001; 276: 3417-3425Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). When this translocating activity is abrogated, cholesterol accumulates in late endosomes and lysosomes in the cells of every organ, including the brain, in proportion to the amount of sterol normally taken up by that tissue through the coated pit pathway (13Xie C. Turley S.D. Dietschy J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11992-11997Crossref PubMed Scopus (83) Google Scholar, 16Xie C. Burns D.K. Turley S.D. Dietschy J.M. J. Neuropathol. Exp. Neurol. 2000; 59: 1106-1117Crossref PubMed Scopus (95) Google Scholar). As a result, the cholesterol pool in the whole mouse progressively expands with age, reaching 2.5 times the normal value at 7 weeks (B1) (17Xie C. Turley S.D. Pentchev P.G. Dietschy J.M. Am. J. Physiol. 1999; 276: E336-E344PubMed Google Scholar). However, functional loss of this sequestered sterol is compensated for by an increase in the rate of sterol synthesis in every organ so that the flux of cholesterol from the extrahepatic tissues to the liver (B2) and the excretion of this sterol in the feces (B4, B5) continues at essentially normal, or even elevated, rates. Thus, loss of function of NPC1 results in an unusual phenotype in the mouse (and child) in which the pool of cellular cholesterol continuously expands with age whereas the centripetal flow of newly synthesized sterol from the extrahepatic tissues to the liver and from the liver into the feces is essentially normal. By administration of the liver X receptor agonist, T0901317, increased expression of several sterol transporters (ABCA1, ABCG1, ABCG5, and ABCG8) was achieved in a second mouse model. ABCA1 is expressed in most tissues and, particularly, in placenta, endocrine glands, intestine, and liver. Recent data suggest that it may play a role in the specific transfer of cholesterol across the plasma membrane of cells to lipid-poor apoA-I particles (18Oram J.F. Vaughn A.M. Curr. Opin. Lipidol. 2000; 11: 253-260Crossref PubMed Scopus (238) Google Scholar, 19Chambenoit O. Hamon Y. Didier M. Rigneault H. Rosseneu M. Chimini G. J. Biol. Chem. 2001; 276: 9955-9960Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 20Oram J.F. Lawn R.M. J. Lipid Res. 2001; 42: 1173-1179Abstract Full Text Full Text PDF PubMed Google Scholar). Certainly, this appears to be the case for cells such as macrophages. However, the question remains as to its role in the steady-state movement of cholesterol from the organs of the extrahepatic compartment to the liver. If this protein were rate-limiting for the translocation of cholesterol between the intracellular pools and the plasma space, then overexpression of ABCA1 should increase sterol turnover in the extrahepatic tissues of the mouse whereas deletion of this activity should markedly suppress extrahepatic cholesterol synthesis and fecal sterol output. The limited data currently available, however, do not support either of these predictions. When ABCA1 is overexpressed pharmacologically, the whole animal sterol pool (C1) and the flux of cholesterol from the extrahepatic tissues to the liver (C2) remain essentially unchanged. There is, however, a 5-fold increase in the rate of hepatic cholesterol synthesis and a corresponding increase in the excretion of fecal neutral, but not acidic, sterols (C4, C5). Similarly, when ABCA1 activity is deleted, the whole mouse cholesterol pool remains constant, and the movement of sterol from the extrahepatic tissues through the liver to the feces also remains essentially unchanged (21McNeish J. Aiello R.J. Guyot D. Turi T. Gabel C. Aldinger C. Hoppe K.L. Roach M.L. Royer L.J. de Wet J. Broccardo C. Chimini G. Francone O.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4245-4250Crossref PubMed Scopus (475) Google Scholar, 22Drobnik W. Lindenthal B. Lieser B. Ritter M. Christiansen Weber T. Liebisch G. Giesa U. Igel M. Borsukova H. Büchler C. Fung-Leung W.P. Gastroenterology. 2001; 120: 1203-1211Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Thus, whereas such balance data are incomplete, those that are available suggest that ABCA1 may be important for the removal of cholesterol from cells like macrophages but not from the majority of the cells making up the tissues of the extrahepatic compartment. Abrogation of the function of specific transport proteins markedly lowers the rate of clearance of cholesterol carried in specific lipoprotein fractions in vivo, as it does in isolated cells studied in vitro. For example, in the LDLR−/−mouse, the clearance of cholesterol carried in LDL by the liver is reduced from a normal value of ∼400 ml/day/kg to only 25 ml/day/kg. Similarly, the hepatic clearance of sterol carried in HDL is reduced from 60 ml/day/kg to only 6 ml/day/kg in the SR-BI−/−animal. However, under these in vivo conditions, the concentration of these two respective lipoproteins increases until the amount of cholesterol removed from the plasma by non-receptor-mediated clearance mechanisms returns to essentially normal, or even elevated, values (10Osono Y. Woollett L.A. Herz J. Dietschy J.M. J. Clin. Invest. 1995; 95: 1124-1132Crossref PubMed Scopus (163) Google Scholar). Thus, there is a marked increase in the plasma cholesterol pool with deletion of function of the LDLR (105 mg/kg, D3), SR-BI (118 mg/kg, E3), and apoE (226 mg/kg, F3) that is attributable to an increase in the concentration of sterol carried in LDL, HDL, and remnant particles, respectively. Despite these changes in the plasma compartment, however, there are virtually no accompanying alterations in the pool of cholesterol in the tissues of the whole animal (D–F1) nor, importantly, is the flux of sterol from the extrahepatic compartment to the liver (D–F2) significantly affected. That net cholesterol balance in these animals is largely unaltered is also supported by the finding that in the mouse (and human) with these deletions, fecal excretion of neutral (D–E4) and acidic (D–F5) sterols is the same as in control animals. Similar findings have been reported with other manipulations affecting the concentration of HDL in the plasma space. The plasma cholesterol pool can be varied 11-fold, from 11 mg/kg in the apoA-I−/− mouse (G3) to 22 mg/kg in the animal and to mg/kg in the mouse in all of these there is in the tissue cholesterol pools is the rate of sterol movement from the extrahepatic compartment to the liver significantly Thus, in deletion or overexpression of the function of proteins involved in the metabolism or transport of specific lipoproteins in the plasma space results in a phenotype with a markedly altered steady-state concentration of these respective lipoproteins in the However, these changes in the plasma cholesterol pool have on the net flux of sterol from the extrahepatic tissues to the liver or from the liver into the fecal neutral and acidic When the rate of bile acid synthesis is significantly reduced by deletion of the activity of or fecal acidic sterol excretion to of the control value Because there is an even greater in the of the pool of bile acid in the small intestine, cholesterol to essentially As a result, there is a marked increase in the loss of fecal neutral sterols that is compensated for by an increase in hepatic synthesis M. D.W. Dietschy J.M. Turley S.D. J. Lipid Res. 1998; Full Text Full Text PDF PubMed Google Scholar, E. D.W. Dietschy J.M. Turley S.D. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Thus, whereas of bile acid synthesis be to cholesterol turnover in the mouse, of this on cholesterol turnover is Similar increases are when are used to the of cholesterol or bile acid from the intestine. of cholesterol is with an increase in fecal neutral but not acidic whereas of bile acid is with an increase in acidic but not neutral fecal Thus, in all of these the has to a change in net sterol balance across the liver with an in cholesterol or bile acid synthesis. In the such changes may be with a in hepatic LDLR activity and a corresponding change in the plasma cholesterol In the mouse, however, these are so that there is usually no change in hepatic LDLR and there is no in the pools of cholesterol in the whole animal or plasma and the flux of sterol from the extrahepatic tissues to the liver is unchanged. In there are and in the of cholesterol balance in the mouse, compared with the Nevertheless, deletion or overexpression of specific protein results in changes in sterol balance that similar changes in humans with or affecting these same In loss of function of proteins such as NPC1 that intracellular cholesterol movement results in of the whole animal sterol pool but no change in the rate of cholesterol movement from the tissues to the liver or from the liver into the In of function of proteins involved in the metabolism of plasma lipoproteins results in changes in the plasma cholesterol pool but not in the of cholesterol in the tissues or in the rate of flow of sterol into the functional changes in the proteins affecting the of cholesterol or bile acid the rates of fecal sterol excretion the pools of cholesterol in the whole animal or