ATP-Citrate Lyase Deficiency in the Mouse
2004; Elsevier BV; Volume: 279; Issue: 10 Linguagem: Inglês
10.1074/jbc.m310512200
ISSN1083-351X
AutoresAnne P. Beigneux, Cynthia Kosinski, Bryant J. Gavino, Jay D. Horton, William C. Skarnes, Stephen G. Young,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoATP-citrate lyase (Acly) is one of two cytosolic enzymes that synthesize acetyl-coenzyme A (CoA). Because acetyl-CoA is an essential building block for cholesterol and triglycerides, Acly has been considered a therapeutic target for hyperlipidemias and obesity. To define the phenotype of Acly-deficient mice, we created Acly knockout mice in which a β-galactosidase marker is expressed from Acly regulatory sequences. We also sought to define the cell type-specific expression patterns of Acly to further elucidate the in vivo roles of the enzyme. Homozygous Acly knockout mice died early in development. Heterozygous mice were healthy, fertile, and normolipidemic on both chow and high fat diets, despite expressing half-normal amounts of Acly mRNA and protein. Fibroblasts and hepatocytes from heterozygous Acly mice contained half-normal amounts of Acly mRNA and protein, but this did not perturb triglyceride and cholesterol synthesis or the expression of lipid biosynthetic genes regulated by sterol regulatory element-binding proteins. The expression of acetyl-CoA synthetase 1, another cytosolic enzyme for producing acetyl-CoA, was not up-regulated. As judged by β-galactosidase staining, Acly was expressed ubiquitously but was expressed particularly highly in tissues with high levels of lipogenesis, such as in the livers of mice fed a high-carbohydrate diet. β-Galactosidase staining was intense in the developing brain, in keeping with the high levels of de novo lipogenesis of the tissue. In the adult brain, β-galactosidase staining was in general much lower, consistent with reduced levels of lipogenesis; however, β-galactosidase expression remained very high in cholinergic neurons, likely reflecting the importance of Acly in generating acetyl-CoA for acetylcholine synthesis. The Acly knockout allele is useful for identifying cell types with a high demand for acetyl-CoA synthesis. ATP-citrate lyase (Acly) is one of two cytosolic enzymes that synthesize acetyl-coenzyme A (CoA). Because acetyl-CoA is an essential building block for cholesterol and triglycerides, Acly has been considered a therapeutic target for hyperlipidemias and obesity. To define the phenotype of Acly-deficient mice, we created Acly knockout mice in which a β-galactosidase marker is expressed from Acly regulatory sequences. We also sought to define the cell type-specific expression patterns of Acly to further elucidate the in vivo roles of the enzyme. Homozygous Acly knockout mice died early in development. Heterozygous mice were healthy, fertile, and normolipidemic on both chow and high fat diets, despite expressing half-normal amounts of Acly mRNA and protein. Fibroblasts and hepatocytes from heterozygous Acly mice contained half-normal amounts of Acly mRNA and protein, but this did not perturb triglyceride and cholesterol synthesis or the expression of lipid biosynthetic genes regulated by sterol regulatory element-binding proteins. The expression of acetyl-CoA synthetase 1, another cytosolic enzyme for producing acetyl-CoA, was not up-regulated. As judged by β-galactosidase staining, Acly was expressed ubiquitously but was expressed particularly highly in tissues with high levels of lipogenesis, such as in the livers of mice fed a high-carbohydrate diet. β-Galactosidase staining was intense in the developing brain, in keeping with the high levels of de novo lipogenesis of the tissue. In the adult brain, β-galactosidase staining was in general much lower, consistent with reduced levels of lipogenesis; however, β-galactosidase expression remained very high in cholinergic neurons, likely reflecting the importance of Acly in generating acetyl-CoA for acetylcholine synthesis. The Acly knockout allele is useful for identifying cell types with a high demand for acetyl-CoA synthesis. ATP-citrate lyase (Acly) 1The abbreviations used are: Acly, ATP-citrate lyase; Acs1, acetyl-CoA synthetase 1; SREBP, sterol regulatory element-binding protein; βgeo, a fusion of β-galactosidase and neomycin phosphotransferase II; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; dpc, days post-coitus. is a cytosolic enzyme that catalyzes the formation of acetyl-coenzyme A (CoA) and oxaloacetate from citrate and CoA, with the hydrolysis of ATP to ADP and phosphate. Acetyl-CoA is the key building block for de novo lipogenesis. Acly is not, however, the sole source of acetyl-CoA in the cytosol. Another enzyme, acetyl-CoA synthetase 1 (Acs1), generates acetyl-CoA from acetate and CoA, with the hydrolysis of ATP to AMP and diphosphate (1Knowles S.E. Jarrett I.G. Filsell O.H. Ballard F.J. Biochem. J. 1974; 142: 401-411Crossref PubMed Scopus (270) Google Scholar). Whether Asc1 might be sufficient in the setting of a genetic deficiency in Acly is not known. The expression of both Acly and Acs1 is controlled by the sterol regulatory element-binding protein (SREBP)-1c, a transcriptional regulator of fatty acid synthesis (2Luong A. Hannah V.C. Brown M.S. Goldstein J.L. J. Biol. Chem. 2000; 275: 26458-26466Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 3Liang G. Yang J. Horton J.D. Hammer R.E. Goldstein J.L. Brown M.S. J. Biol. Chem. 2002; 277: 9520-9528Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar). An important role for Acly in lipid biosynthesis in the liver has been suggested by studies with the Acly inhibitor hydroxycitrate. In rats treated with hydroxycitrate, the rate of fatty acid and cholesterol synthesis in the liver fall (4Sullivan A.C. Triscari J. Hamilton J.G. Miller O.N. Wheatley V.R. Lipids. 1974; 9: 121-128Crossref PubMed Scopus (115) Google Scholar). Those experiments and studies with other inhibitors (5Bray G.A. Greenway F.L. Clin. Endocrinol. Metab. 1976; 5: 455-479Abstract Full Text PDF PubMed Scopus (13) Google Scholar, 6Chee H. Romsos D.R. Leveille G.A. J. Nutr. 1977; 107: 112-119Crossref PubMed Scopus (15) Google Scholar, 7Greenwood M.R. Cleary M.P. Gruen R. Blase D. Stern J.S. Triscari J. Sullivan A.C. Am. J. Physiol. 1981; 240: E72-E78PubMed Google Scholar, 8Saxty B.A. Novelli R. Dolle R.E. Kruse L.I. Reid D.G. Camilleri P. Wells T.N. Eur. J. Biochem. 1991; 202: 889-896Crossref PubMed Scopus (16) Google Scholar, 9Pearce N.J. Yates J.W. Berkhout T.A. Jackson B. Tew D. Boyd H. Camilleri P. Sweeney P. Gribble A.D. Shaw A. Groot P.H. Biochem. J. 1998; 334: 113-119Crossref PubMed Scopus (109) Google Scholar, 10Leonhardt M. Hrupka B. Langhans W. Physiol. Behav. 2001; 74: 191-196Crossref PubMed Scopus (51) Google Scholar) have suggested that Acly could represent a therapeutic target for elevated plasma lipid levels. As with other genes involved in lipid synthesis, the expression of Acly in the liver is regulated, at least in part, at the transcriptional level by SREBP-1 (3Liang G. Yang J. Horton J.D. Hammer R.E. Goldstein J.L. Brown M.S. J. Biol. Chem. 2002; 277: 9520-9528Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar, 11Shimomura I. Shimano H. Korn B.S. Bashmakov Y. Horton J.D. J. Biol. Chem. 1998; 273: 35299-35306Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 12Shimano H. Yahagi N. Amemiya-Kudo M. Hasty A.H. Osuga J. Tamura Y. Shionoiri F. Iizuka Y. Ohashi K. Harada K. Gotoda T. Ishibashi S. Yamada N. J. Biol. Chem. 1999; 274: 35832-35839Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar). When lipid biosynthesis levels in the liver are low, as in fasted mice, Acly mRNA levels are low. However, when fasted mice are fed a carbohydrate-rich diet, lipid biosynthesis rates and Acly mRNA levels increase (3Liang G. Yang J. Horton J.D. Hammer R.E. Goldstein J.L. Brown M.S. J. Biol. Chem. 2002; 277: 9520-9528Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar, 12Shimano H. Yahagi N. Amemiya-Kudo M. Hasty A.H. Osuga J. Tamura Y. Shionoiri F. Iizuka Y. Ohashi K. Harada K. Gotoda T. Ishibashi S. Yamada N. J. Biol. Chem. 1999; 274: 35832-35839Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar). Lipid synthesis is important in the developing brain, particularly for myelination (13Dietschy J.M. Turley S.D. Curr. Opin. Lipidol. 2001; 12: 105-112Crossref PubMed Scopus (733) Google Scholar). Northern blots have shown that Acly mRNA levels increase in the rat brain during the first week of suckling, when de novo lipogenesis is high (14Elshourbagy N.A. Near J.C. Kmetz P.J. Sathe G.M. Southan C. Strickler J.E. Gross M. Young J.F. Wells T.N. Groot P.H. J. Biol. Chem. 1990; 265: 1430-1435Abstract Full Text PDF PubMed Google Scholar). However, those experiments have not revealed which regions of the brain, or which cell types in the brain, express Acly at high levels. Acly is also highly expressed in the kidney (15Foster D.W. Srere P.A. J. Biol. Chem. 1968; 243: 1926-1930Abstract Full Text PDF PubMed Google Scholar, 16Melnick J.Z. Srere P.A. Elshourbagy N.A. Moe O.W. Preisig P.A. Alpern R.J. J. Clin. Invest. 1996; 98: 2381-2387Crossref PubMed Scopus (79) Google Scholar), and moderate amounts of the Acly mRNA are found in the adrenals, intestine, and lung (14Elshourbagy N.A. Near J.C. Kmetz P.J. Sathe G.M. Southan C. Strickler J.E. Gross M. Young J.F. Wells T.N. Groot P.H. J. Biol. Chem. 1990; 265: 1430-1435Abstract Full Text PDF PubMed Google Scholar). In this study, we explored the physiologic importance of Acly in mice. We had three goals. First, we were interested in determining if a complete absence of Acly would be compatible with embryonic development. Second, we wanted to determine whether half-normal levels of Acly, as in heterozygous Acly knockout mice, would be associated with alterations in lipid synthesis, plasma lipid levels, or body weight. Third, we sought to define the cell type-specific expression of Acly, both during development and in adult mice, to better understand the in vivo roles of Acly. To address these questions, we produced and characterized Acly-deficient mice. Acly-deficient Mice—A mouse embryonic stem cell line (cell line NPX098, strain 129/Ola) containing an insertional mutation in Acly was identified within BayGenomics, a gene-trapping resource (17Stryke D. Kawamoto M. Huang C.C. Johns S.J. King L.A. Harper C.A. Meng E.C. Lee R.E. Yee A. L'Italien L. Chuang P.T. Young S.G. Skarnes W.C. Babbitt P.C. Ferrin T.E. Nucleic Acids Res. 2003; 31: 278-281Crossref PubMed Scopus (209) Google Scholar). The gene-trap vector used (pGT1δTMpfs) contains a splice-acceptor sequence upstream of a reporter gene, βgeo (a fusion of β-galactosidase and neomycin phosphotransferase II) (17Stryke D. Kawamoto M. Huang C.C. Johns S.J. King L.A. Harper C.A. Meng E.C. Lee R.E. Yee A. L'Italien L. Chuang P.T. Young S.G. Skarnes W.C. Babbitt P.C. Ferrin T.E. Nucleic Acids Res. 2003; 31: 278-281Crossref PubMed Scopus (209) Google Scholar). As judged by 5′ rapid amplification of cDNA ends (18Townley D.J. Avery B.J. Rosen B. Skarnes W.C. Genome Res. 1997; 7: 293-298Crossref PubMed Scopus (72) Google Scholar), the insertional mutation in NPX098 occurred in the first intron of Acly, following a noncoding first exon. Thus, the mutation results in the production of a fusion transcript consisting of exon 1 sequences from Acly and βgeo. The mutation created a new EcoRV site in intron 1, which was useful for genotyping mice by Southern blots (see below). NPX098 was injected into C57BL/6 blastocysts to generate chimeric mice, which were bred to establish Acly knockout mice. All mice described herein had a mixed genetic background (∼50% C57BL/6 and ∼50% 129/Ola). The mice were weaned at 21 days of age, housed in a barrier facility with a 12-h light/12-h dark cycle, and fed a chow diet containing 4.5% fat (Ralston Purina, St. Louis, MO). In some experiments, the mice were fed a Western diet (21% fat, 50% carbohydrate, and 20% protein) from Research Diets (New Brunswick, NJ) or a fat-free, carbohydrate-rich diet (60.2% sucrose and 20% casein) from ICN (Irvine, CA). Mice were genotyped by Southern blots with EcoRV-digested genomic DNA and a 5′ Acly flanking probe. The probe was amplified from mouse genomic DNA with primers 5′-CGCTGGTCAAGAATGGGTGTTACAA-3′ and 5′-CTCCCTCCCCCAACCTCAAACTAAG-3′. Mouse Primary Embryonic Fibroblasts—Timed matings were established between Acly+/- mice, and pregnant females were sacrificed 13.5-17.5 days post-coitus (dpc). Embryos were incubated overnight in 5 ml of 0.25% trypsin-EDTA (Invitrogen, Carslbad, CA) at 4 °C. The next morning, embryos were mechanically disrupted in 5 ml of Dulbecco’s modified Eagle’s medium supplemented with fetal bovine serum, l-glutamine, nonessential amino acids, penicillin, streptomycin, Fungizone (Invitrogen), and 2-mercaptoethanol (Sigma). After removal of debris, cells were plated in 100-mm Petri dishes. Mouse Primary Hepatocytes—Mice were anesthetized with 2.5% avertin, and a catheter was introduced into the inferior vena cava via the right atrium. The liver was perfused with liver perfusion media (Invitrogen) and then collagenase-lipase media (Invitrogen). The digested liver was then removed and mechanically disrupted. The cell suspension was filtered through sterile gauze and centrifuged at 50 × g. Hepatocytes were then washed three times with hepatocyte wash media (Invitrogen), and 5 × 105 cells/well were plated in 6-well collagen-coated tissue culture plates. Cells were allowed to attach to the plate for 4 h before starting the labeling experiments (see below). Cells were grown at 37 °C under 5% CO2 in 3 ml of Dulbecco’s modified Eagle’s medium supplemented with fetal bovine serum, sodium pyruvate, l-glutamine, nonessential amino acids, penicillin, streptomycin, and Fungizone. Analysis of Cholesterol and Triglyceride Synthesis by Thin-layer Chromatography—Acly+/+ and Acly+/- fibroblasts were grown to 90% confluency in 100-mm Petri dishes. Cells were then labeled with 50 μCi of [1(3)-3H]glycerol (Amersham Biosciences), 10 μCi of [1,5-14C]citric acid (American Radiolabeled Chemicals, St. Louis, MO), or 25 μCi of [1-14C]acetic acid (Amersham Biosciences). Primary hepatocytes were labeled with 10 μCi of [1(3)-3H]glycerol. Lipids from fibroblasts and hepatocytes were extracted with hexane:isopropyl alcohol (3:2 (v/v)), dried under nitrogen, and resuspended in 100 μl of chloroform. Triglycerides and free cholesterol were separated on thin-layer chromatography silica plates (60 Å) in hexane:ethyl ether:acetic acid (80:20:1 (v/v/v)). Samples were assayed in duplicates (40 μl/lane). After lipid extraction, cell protein was quantified with the Bradford assay (Bio-Rad). Blood samples were taken from the retroorbital sinus under anesthesia (2.5% avertin). Plasma cholesterol, triglycerides, and free fatty acids were measured with enzymatic assays (Abbott Spectrum (Abbott, Abbott Park, IL), triglycerides/glycerol blanked (Roche/Hitachi, Indianapolis, IN), and free fatty acids half-micro test (Roche Applied Science), respectively). Liver lipids were extracted (19Björkegren J. Beigneux A. Bergo M.O. Maher J.J. Young S.G. J. Biol. Chem. 2002; 277: 5476-5483Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), and the amounts of cholesterol and triglycerides were measured (20Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M.S. Goldstein J.L. J. Clin. Invest. 1996; 98: 1575-1584Crossref PubMed Scopus (698) Google Scholar). Before harvesting of liver tissue, mice were anesthetized and perfused with saline. RNA Isolation and Northern Blot Analysis—Total RNA was isolated from 50 to 150 mg of mouse tissue or from fibroblasts grown on 100-mm Petri dishes with Tri-Reagent (Sigma). Total RNA (25 μg) was separated by electrophoresis on a 1% agarose/formaldehyde gel and transferred onto a Nytran SuPerCharge membrane (Schleicher & Schuell). A mouse multiple-tissue poly(A)+ RNA blot and a mouse embryo poly(A)+ RNA blot (Clontech, Palo Alto, CA) were used to determine the tissue pattern of Acly expression in adult mice and to examine the temporal expression of Acly during embryogenesis. Bands on Northern blots were visualized by autoradiography (Hyperfilm ECL, Amersham Biosciences) and quantified by densitometry (Molecular Imager FX, Bio-Rad). We used the same Acly cDNA probe that has been described previously (11Shimomura I. Shimano H. Korn B.S. Bashmakov Y. Horton J.D. J. Biol. Chem. 1998; 273: 35299-35306Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). A β-galactosidase cDNA was amplified by reverse transcriptase-PCR from Acly+/- mouse liver RNA with primers 5′-TTTTCGATGAGCGTGGTGGTTATGC-3′ and 5′-GCGCGTACATCGGGCAAATAATATC-3′; an Asc1 cDNA was amplified with oligonucleotides 5′-TGCTGAGGACCCACTCTTCATCTTG-3′ and 5′-AAGCAGAACCAGGTTTCATGGGTGT-3′. A glyceraldehyde-3-phosphate dehydrogenase (Gapdh) probe and an 18 S cDNA were used as controls for RNA integrity and loading. [α-32P]dCTP-labeled cDNA probes were prepared with All-in-One random prime labeling mixture (Sigma). Standard prehybridization, hybridization, and washing procedures were used (21Beigneux A. Withycombe S.K. Digits J.A. Tschantz W.R. Weinbaum C.A. Griffey S.M. Bergo M. Casey P.J. Young S.G. J. Biol. Chem. 2002; 277: 38358-38363Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Quantitative Real-time PCR—Real-time PCR conditions and oligonucleotides to measure mRNA levels for Acly, Acs2, acetyl-CoA carboxylase, fatty acid synthase, stearoyl-CoA desaturase 1, glycerol-3-phosphate-acyltransferase, long-chain fatty acyl elongase, glucose-6-phosphatase, glucokinase, glucose-6-phosphate dehydrogenase, malic enzyme, 3-hydroxy-3-methylglutaryl-CoA synthase, and 3-hydroxy-3-methylglutaryl-CoA reductase were described previously (22Moon Y.A. Horton J.D. J. Biol. Chem. 2003; 278: 7335-7343Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). The level of Gapdh mRNA was used as a control. Western Blot Analysis—Fresh mouse tissues (liver, white adipose tissue, kidney, and brain) (0.5-1 g), or embryonic fibroblasts (grown to 80-90% confluency in 100-mm Petri dishes) were homogenized in RIPA buffer containing a mixture of protease inhibitors (Complete Mini EDTA-free, Roche Applied Science). Samples were incubated on ice for 30 min and centrifuged at 14,000 rpm for 10 min at 4 °C. The protein content of the supernatant fluid was determined with the Bradford assay (Bio-Rad). Denatured proteins (60-100 μg) were loaded onto precast 4-15% polyacrylamide gels (Bio-Rad). After electrotransfer onto polyvinylidene difluoride membrane (Amersham Biosciences), blots were blocked with phosphate-buffered saline containing 0.1% Tween and 3% bovine serum albumin for 4 h at 4 °C and incubated for 1 h at room temperature with a rabbit antibody against rat Acly (14Elshourbagy N.A. Near J.C. Kmetz P.J. Sathe G.M. Southan C. Strickler J.E. Gross M. Young J.F. Wells T.N. Groot P.H. J. Biol. Chem. 1990; 265: 1430-1435Abstract Full Text PDF PubMed Google Scholar) at a dilution of 1:500 in phosphate-buffered saline containing 0.10% bovine serum albumin. Blots were then incubated with a horseradish peroxidase-linked donkey anti-rabbit IgG antibody (Amersham Biosciences) at a dilution of 1:50,000 in phosphate-buffered saline containing 0.1% bovine serum albumin for 45 min at room temperature. Antibody binding was detected with the ECL Plus Western blotting kit (Amersham Biosciences); the blots were exposed to Hyperfilm ECL (Amersham Biosciences) and quantified by densitometry as described above. Immunohistochemistry and β-Galactosidase Staining—Mice were anesthetized with avertin and perfusion-fixed with 4% paraformaldehyde. Tissues were harvested and fixed in 10% formalin for 4 h at 4 °C, immersed in 30% glucose for ∼16 h at 4 °C, and frozen in O.C.T. (Sakura Finetek, Torrance, CA) for cryostat sectioning. β-Galactosidase activity was assessed by incubating 10-μm thick sections with a staining solution containing 1.3 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) (Invitrogen) for 16 h at 37 °C (23Bergo M.O. Gavino B.J. Steenbergen R. Sturbois B. Parlow A.F. Sanan D.A. Skarnes W.C. Vance J.E. Young S.G. J. Biol. Chem. 2002; 277: 47701-47708Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). After staining, slides were counterstained with Eosin Y (Sigma). In some experiments, β-galactosidase staining was followed by immunohistochemistry. The following primary antibodies were used: rabbit anti-human glial fibrillary acidic protein (Sigma, 1:1,000), mouse monoclonal anti-neuronal nuclei (Chemicon, Temecula, CA, 1:1,000), goat anti-human choline acetyltransferase (Chemicon, 1:1,000), and goat anti-rat vesicular acetylcholine transporter (Chemicon, 1:5,000). Sections were subsequently incubated with the appropriate biotinylated secondary antibody (anti-rabbit or anti-goat IgG), and antigen-antibody complexes were detected after incubation with horseradish peroxidase and diaminobenzidine substrate (ABC kit, Vector Laboratories, Burlingame, CA). Sections were examined and photographed on an Eclipse 6600 microscope (Nikon, Japan) equipped with a Spot RT Slider digital camera (Diagnostic Instruments, Burlingame, CA). The insertional mutation in the first intron of Acly introduced a new EcoRV site, making it possible to use Southern blots to distinguish between a 29.2-kilobase (kb) EcoRV fragment in the wild-type allele and a shorter (∼6.2-kb) EcoRV fragment in the mutant allele (Fig. 1A). The mutation results in the production of a fusion transcript containing sequences from exon 1 joined to βgeo (Fig. 1B). There is little doubt that the insertional mutation inactivated Acly. Acly mRNA levels were decreased by ∼50% in Acly+/- mice (Fig. 1B), as were Acly protein levels (Fig. 1C). Acly+/- mice were intercrossed in an attempt to obtain homozygous knockout mice. However, genotyping of more than 60 litters did not yield any homozygous mice. Moreover, we genotyped more than 80 embryos from 8.5 to 17.5 dpc, and found no homozygous knockout embryos. Acly is expressed at high levels throughout embryonic development (Fig. 2A), and β-galactosidase staining revealed that Acly is expressed at high levels in the neural tube at 8.5 dpc (Fig. 2B). We concluded that Acly expression in embryos is required for development. We predicted that Acly+/- mice would have reduced body weight and lower plasma lipid levels as a consequence of a reduced capability for lipid synthesis. However, the levels of cholesterol, triglycerides, and free fatty acids in the plasma were no different in chow-fed Acly+/+ and Acly+/- mice (Table I). Moreover, feeding the mice a Western diet did not yield any significant difference in plasma lipid levels (Table I) or in hepatic cholesterol and triglyceride levels (not shown). Body weights of Acly+/+ and Acly+/- mice were virtually identical (Table I).Table IPlasma levels of cholesterol, triglycerides, and free fatty acids (FFA) in Acly +/+ and Acly +/− mice fed a Western dietBaseline1 month+/+ (n = 18)+/− (n = 14)+/+ (n = 18)+/− (n = 14)Body weight (g)20.6 ± 0.6319.1 ± 1.0733.0 ± 0.7031.1 ± 1.13Cholesterol (mg/dl)83.4 ± 3.4182.1 ± 3.61148 ± 9.28137 ± 8.34Triglycerides (mg/dl)67.4 ± 18.452.4 ± 14.6129 ± 18.7113 ± 14.2FFA (μm)411 ± 33.7428 ± 70.6688 ± 78.7668 ± 106 Open table in a new tab We suspected that Acly+/- mice might exhibit reduced weight gain and lower plasma lipid levels when fed a high-carbohydrate diet, which results in high levels of de novo lipogenesis in the liver. Contrary to our hypothesis, however, body weights and plasma levels of cholesterol, triglycerides, free fatty acids, and glucose were similar in Acly+/+ and Acly+/- mice over 3 months of observation (Table II). Also, feeding the carbohydrate-rich diet to Acly+/- mice was not associated with reduced liver stores of cholesterol and triglycerides (Table III).Table IIPlasma levels of cholesterol, triglycerides, and free fatty acids (FFA) in Acly +/+ and Acly +/− mice fed a fat-free, carbohydrate-rich dietBaseline1 month+/+ (n = 8)+/− (n = 8)+/+ (n = 8)+/− (n = 8)Body weight (g)21.2 ± 0.8018.5 ± 1.2629.2 ± 1.2426.4 ± 1.31Cholesterol (mg/dl)95.3 ± 5.0085.3 ± 3.8883.3 ± 16.478.8 ± 3.78Triglycerides (mg/dl)49.8 ± 8.2542.6 ± 6.2112.8 ± 2.3714.1 ± 2.55FFA (μm)435 ± 50.1428 ± 70.7413 ± 61.4318 ± 39.7 Open table in a new tab Table IIIHepatic cholesterol and triglyceride contents in Acly +/+ and Acly +/− mice fed a fat-free, carbohydrate-rich dietBaseline3 months+/+ (n = 6)+/− (n = 14)+/+ (n = 6)+/− (n = 14)Cholesterol (mg/g protein)7.88 ± 1.165.97 ± 0.7732.3 ± 17.049.1 ± 7.21Triglycerides (mg/g protein)395 ± 39.0361 ± 19.1702 ± 207628 ± 53.2 Open table in a new tab We also examined lipid synthesis in primary fibroblasts from Acly+/+ and Acly+/- embryos. As expected, Acly mRNA (Fig. 3A) and protein levels (Fig. 3B) were reduced by ∼50% in heterozygous cells. However, the amount of cholesterol synthesis in Acly+/+ and Acly+/- fibroblasts was similar, as judged by [14C]citrate metabolic labeling experiments (Fig. 3C). It was not possible to assess the impact of reduced Acly expression on triglyceride synthesis, because very little [14C]citrate was incorporated into triglycerides. However, Acly+/+ and Acly+/- fibroblasts displayed similar amounts of triglyceride synthesis in [3H]glycerol labeling experiments (Fig. 3C). Similarly, cholesterol and triglyceride synthesis rates were entirely normal in primary hepatocytes from Acly+/- mice (Fig. 4).Fig. 4Levels of cholesterol and triglyceride synthesis in primary hepatocytes from Acly+/+ and Acly+/- mice. Cholesterol and triglyceride synthesis rates in Acly+/- cells are expressed as a percentage of those in Acly+/+ cells. The levels of incorporation of [1(3)-3H]glycerol into cholesterol and triglycerides in wild-type cells were 583 ± 33 cpm/μg of protein and 56,085 ± 2,919 cpm/μg of protein, respectively. Data represent the average of triplicate determinations, and each experiment was repeated twice.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The fact that the Acly+/- mice and Acly+/- cells did not manifest detectable perturbations in lipid levels or lipid biosynthesis suggested that half-normal levels of Acly have little impact on cellular lipid homeostasis. Indeed, this appeared to be the case. As judged by quantitative reverse transcriptase-PCR, Acly mRNA levels were reduced by 50% in Acly+/- cells, but the expression of a host of other enzymes involved in triglyceride, cholesterol, and glucose metabolism was not perturbed (Figs. 3D and 5A). Of note, the half-normal levels of Acly expression did not lead to an up-regulation of the expression of Asc1, the other acetyl-CoA-synthesizing enzyme in the cytosol (Figs. 3A and 5B). In keeping with the latter finding, the synthesis of cholesterol and triglycerides from [14C]acetate (a measure of Acs1 activity (2Luong A. Hannah V.C. Brown M.S. Goldstein J.L. J. Biol. Chem. 2000; 275: 26458-26466Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar)) was identical in Acly+/+ and Acly+/- cells (Fig. 3C). Northern blots showed that Acly is expressed in multiple tissues (Fig. 6A). To explore the cell-type specific expression patterns, sections of tissues from Acly+/- mice were stained for β-galactosidase expression. Strong β-galactosidase expression was identified in multiple cell types, including Leydig cells in testis (Fig. 6B) and renal tubular cells (Fig. 6C). In the livers of chow-fed mice, β-galactosidase staining was largely confined to the periportal hepatocytes (Fig. 6D). In mice fed a fat-free, carbohydrate-rich diet, β-galactosidase staining of the liver was much more widespread and more intense (Fig. 6E), reflecting increased levels of Acly expression (Fig. 6G). On the Western diet, β-galactosidase staining of the liver was only modestly increased, compared with the chow diet (Fig. 6F). β-Galactosidase expression was intense throughout the brains of 15.5 dpc Acly+/- embryos (Fig. 7A) and 1-day-old Acly+/- pups (Fig. 7B), consistent with the presumed role of Acly in the de novo lipogenesis required for myelin formation (14Elshourbagy N.A. Near J.C. Kmetz P.J. Sathe G.M. Southan C. Strickler J.E. Gross M. Young J.F. Wells T.N. Groot P.H. J. Biol. Chem. 1990; 265: 1430-1435Abstract Full Text PDF PubMed Google Scholar). We suspected that Acly expression in the brain would decrease in older animals, mirroring a reduced requirement for lipid synthesis (24Spady D.K. Dietschy J.M. J. Lipid Res. 1983; 24: 303-315Abstract Full Text PDF PubMed Google Scholar, 25Dietschy J.M. Kita T. Suckling K.E. Goldstein J.L. Brown M.S. J. Lipid Res. 1983; 24: 469-480Abstract Full Text PDF PubMed Google Scholar, 26Jurevics H. Morell P. J. Neurochem. 1995; 64: 895-901Crossref PubMed Scopus (250) Google Scholar, 27Jurevics H.A. Kidwai F.Z. Morell P. J. Lipid Res. 1997; 38: 723-733Abstract Full Text PDF PubMed Google Scholar, 28Turley S.D. Burns D.K. Dietschy J.M. Am. J. Physiol. 1998; 274: E1099-E1105Crossref PubMed Google Scholar). This suspicion was confirmed. In 3-week-old pups, β-galactosidase staining was less intense than in the 1-day-old mice, although staining was still observed in the hippocampus, thalamus, medulla, and the white matter of cerebellum (Fig. 7C). In 6-month-old mice, Acly was expressed at far lower levels in the brain than in the younger mice (Fig. 7D). In the adult mice, β-galactosidase was expressed in glial cells (Fig. 8A) and in neurons (Fig. 8B).Fig. 8Acly is expressed in glial cells and in neurons in the adult mouse nervous system.A and B, β-galactosidase and immunohistochemical staining of adult Acly+/- thalamus (A) and cortex (B). Specific antibodies against glial fibrillary acidic protein (A) and neurona
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