Cloning and characterization of a mammalian fatty acyl-CoA elongase as a lipogenic enzyme regulated by SREBPs
2002; Elsevier BV; Volume: 43; Issue: 6 Linguagem: Inglês
10.1016/s0022-2275(20)30465-x
ISSN1539-7262
AutoresTakashi Matsuzaka, Hitoshi Shimano, Naoya Yahagi, Tomohiro Yoshikawa, Michiyo Amemiya-Kudo, Alyssa H. Hasty, Hiroaki Okazaki, Yoshiaki Tamura, Yoko Iizuka, Ken Ohashi, Jun-ichi Osuga, Akimitsu Takahashi, Shigeru Yato, Hirohito Sone, Shun Ishibashi, Nobuhiro Yamada,
Tópico(s)Lipid metabolism and biosynthesis
ResumoThe mammalian enzyme involved in the final elongation of de novo fatty acid biosynthesis following the building of fatty acids to 16 carbons by fatty acid synthase has yet to be identified. In the process of searching for genes activated by sterol regulatory element-binding protein 1 (SREBP-1) by using DNA microarray, we identified and characterized a murine cDNA clone that is highly similar to a fatty acyl-CoA elongase gene family such as Cig30, Sscs, and yeast ELOs. Studies on the cells overexpressing the full-length cDNA indicate that the encoded protein, designated fatty acyl-CoA elongase (FACE), has a FACE activity specific for long-chains; C12-C16 saturated- and monosaturated-fatty acids. Hepatic expression of this identified gene was consistently activated in the livers of transgenic mice overexpressing nuclear SREBP-1a, -1c, or -2. FACE mRNA levels are markedly induced in a refed state after fasting in the liver and adipose tissue. This refeeding response is significantly reduced in SREBP-1 deficient mice. Dietary PUFAs caused a profound suppression of this gene expression, which could be restored by SREBP-1c overexpression. Hepatic FACE expression was also highly up-regulated in leptin-deficient ob/ob mice. Hepatic FACE mRNA was markedly increased by administration of a pharmacological agonist of liver X-activated receptor (LXR), a dominant activator for SREBP-1c expression.These data indicated that this elongase is a new member of mammalian lipogenic enzymes regulated by SREBP-1, playing an important role in de novo synthesis of long-chain saturated and monosaturated fatty acids in conjunction with fatty acid synthase and stearoyl-CoA desaturase. The mammalian enzyme involved in the final elongation of de novo fatty acid biosynthesis following the building of fatty acids to 16 carbons by fatty acid synthase has yet to be identified. In the process of searching for genes activated by sterol regulatory element-binding protein 1 (SREBP-1) by using DNA microarray, we identified and characterized a murine cDNA clone that is highly similar to a fatty acyl-CoA elongase gene family such as Cig30, Sscs, and yeast ELOs. Studies on the cells overexpressing the full-length cDNA indicate that the encoded protein, designated fatty acyl-CoA elongase (FACE), has a FACE activity specific for long-chains; C12-C16 saturated- and monosaturated-fatty acids. Hepatic expression of this identified gene was consistently activated in the livers of transgenic mice overexpressing nuclear SREBP-1a, -1c, or -2. FACE mRNA levels are markedly induced in a refed state after fasting in the liver and adipose tissue. This refeeding response is significantly reduced in SREBP-1 deficient mice. Dietary PUFAs caused a profound suppression of this gene expression, which could be restored by SREBP-1c overexpression. Hepatic FACE expression was also highly up-regulated in leptin-deficient ob/ob mice. Hepatic FACE mRNA was markedly increased by administration of a pharmacological agonist of liver X-activated receptor (LXR), a dominant activator for SREBP-1c expression. These data indicated that this elongase is a new member of mammalian lipogenic enzymes regulated by SREBP-1, playing an important role in de novo synthesis of long-chain saturated and monosaturated fatty acids in conjunction with fatty acid synthase and stearoyl-CoA desaturase. Biosynthesis of fatty acids is the major function part of lipogenesis in its role as an energy storage system. Fatty acids with lengths of 16–18 carbon atoms, constituting the majority of total fatty acids in the cells, are major products of de novo synthesis in most mammalian tissues. These long chain fatty acids play an important role in cellular biological functions, including: energy metabolism, membrane fluidity, and others. There appear to be several distinct metabolic pathways that produce long chain fatty acids. Cytoplasmic fatty acid synthase (FAS) plays a major role in the de novo synthesis of fatty acids. However, the elongation of fatty acids by this enzyme terminates at palmitic acid (C16:0). The end product of mammalian lipogenesis is usually oleic acid (C18:1n-9) or vaccenic acid (C18:1n-7) (1Nelson G.J. Kelley D.S. Hunt J.E. Effect of nutritional status on the fatty acid composition of rat liver and cultured hepatocytes..Lipids. 1986; 21: 454-459Crossref PubMed Scopus (29) Google Scholar, 2Ntambi J.M. Regulation of stearoyl-CoA desaturase by polyunsaturated fatty acids and cholesterol..J. Lipid Res. 1999; 40: 1549-1558Abstract Full Text Full Text PDF PubMed Google Scholar). Mammals have long been thought to possess a membrane bound enzyme that elongates and/or desaturates saturated fatty acyl-CoAs produced by FAS or derived from dietary resources (3Cinti D.L. Cook L. Nagi M.N. Suneja S.K. The fatty acid chain elongation system of mammalian endoplasmic reticulum..Prog. Lipid Res. 1992; 31: 1-51Crossref PubMed Scopus (192) Google Scholar). Stearoyl-CoA desaturase (SCD) has been shown to be committed to the desaturation; however, the gene catalyzing for the C2 elongation of the C16:0 and C16:1 has never been identified. To date, several enzymes involved in the elongation of long-chain fatty acids in non-mammalian cells have been identified. One such family consists of the yeast ELO genes. The yeast ELO1 gene is involved in the elongation of C14:0 to C16:0 (4Toke D.A. Martin C.E. Isolation and characterization of a gene affecting fatty acid elongation in Saccharomyces cerevisiae..J. Biol. Chem. 1996; 271: 18413-18422Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). The ELO2 and ELO3 genes were identified based on the homology to the ELO1 gene (5Oh C.S. Toke D.A. Mandala S. Martin C.E. ELO2 and ELO3, homologues of the Saccharomyces cerevisiae ELO1 gene, function in fatty acid elongation and are required for sphingolipid formation..J. Biol. Chem. 1997; 272: 17376-17384Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar). ELO2 protein is involved in the elongation of saturated and monounsaturated fatty acids up to 24 carbons in length, while ELO3 elongates a broader group of saturated and monounsaturated fatty acids, and is essential for the conversion of C24:0 into C26:0 (5Oh C.S. Toke D.A. Mandala S. Martin C.E. ELO2 and ELO3, homologues of the Saccharomyces cerevisiae ELO1 gene, function in fatty acid elongation and are required for sphingolipid formation..J. Biol. Chem. 1997; 272: 17376-17384Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar). Another known elongase family, Cig30, which was originally identified as a cold-induced gene in brown fat, is the first mouse fatty acid elongase identified (6Tvrdik P. Asadi A. Kozak L.P. Nedergaard J. Cannon B. Jacobsson A. Cig30, a mouse member of a novel membrane protein gene family, is involved in the recruitment of brown adipose tissue..J. Biol. Chem. 1997; 272: 31738-31746Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Ssc1 and Ssc2 were cloned based on the homology to Cig30 (7Tvrdik P. Westerberg R. Silve S. Asadi A. Jakobsson A. Cannon B. Loison G. Jacobsson A. Role of a new mammalian gene family in the biosynthesis of very long chain fatty acids and sphingolipids..J. Cell Biol. 2000; 149: 707-717Crossref PubMed Scopus (182) Google Scholar). Complementation studies in yeast mutants indicated that Cig30 and SSC1 are functionally orthologous to ELO2 and ELO3, respectively. The specific activity of Ssc2 has not been identified. Elovl4, a retinal photoreceptor-specific gene, plays a role in the elongation of very long chain fatty acids and has been reported to be a causative gene for inherited macular degeneration (8Zhang K. Kniazeva M. Han M. Li W. Yu Z. Yang Z. Li Y. Metzker M.L. Allikmets R. Zack D.J. Kakuk L.E. Lagali P.S. Wong P.W. MacDonald I.M. Sieving P.A. Figueroa D.J. Austin C.P. Gould R.J. Ayyagari R. Petrukhin K. A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy..Nat. Genet. 2001; 27: 89-93Crossref PubMed Scopus (375) Google Scholar). HELO1, as a member of HELO family cloned by homology to ELO2, is involved in the elongation of PUFAs and monounsaturated fatty acids, whereas the specific activity of HELO2 has not been not identified (9Leonard A.E. Bobik E.G. Dorado J. Kroeger P.E. Chuang L.T. Thurmond J.M. Parker-Barnes J.M. Das T. Huang Y.S. Mukerji P. Cloning of a human cDNA encoding a novel enzyme involved in the elongation of long-chain polyunsaturated fatty acids..Biochem. J. 2000; 350: 765-770Crossref PubMed Scopus (0) Google Scholar). These elongase could consist of a family that share a common structure of at least five membrane-spanning regions and a single histidine-box motif. Sterol regulatory element-binding proteins (SREBPs) are membrane-bound transcription factors that belong to the basic helix-loop-helix leucine zipper family (10Brown M.S. Goldstein J.L. A proteolytic pathway that controls the cholesterol content of membranes, cells, and blood..Proc. Natl. Acad. Sci. USA. 1999; 96: 11041-11048Crossref PubMed Scopus (1103) Google Scholar, 11Brown M.S. Goldstein J.L. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor..Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2980) Google Scholar, 12Brown M.S. Ye J. Rawson R.B. Goldstein J.L. Regulated intramembrane proteolysis: a control mechanism conserved from bacteria to humans..Cell. 2000; 100: 391-398Abstract Full Text Full Text PDF PubMed Scopus (1148) Google Scholar). SREBPs have been established as a regulator for biosynthesis of both cholesterol and fatty acids. To exert transcriptional activity on SRE-containing SREBP target genes, SREBPs have to undergo proteolytic cleavage in a complex with a sterol-sensing cofactor, SREBP-cleavage activating protein, that escorts SREBP for a rER-Golgi trafficking, a key step for regulation of cellular cholesterol biosynthesis. There are three isoforms of SREBP that have been characterized, SREBP-1a and -1c (also known as ADD1), and SREBP-2 (13Yokoyama C. Wang X. Briggs M.R. Admon A. Wu J. Hua X. Goldstein J.L. Brown M.S. SREBP-1, a basic-helix-loop-helix-leucine zipper protein that controls transcription of the low density lipoprotein receptor gene..Cell. 1993; 75: 187-197Abstract Full Text PDF PubMed Scopus (788) Google Scholar, 14Tontonoz P. Kim J.B. Graves R.A. Spiegelman B.M. ADD1: a novel helix-loop-helix transcription factor associated with adipocyte determination and differentiation..Mol. Cell. Biol. 1993; 13: 4753-4759Crossref PubMed Scopus (534) Google Scholar, 15Hua X. Yokoyama C. Wu J. Briggs M.R. Brown M.S. Goldstein J.L. Wang X. SREBP-2, a second basic-helix-loop-helix-leucine zipper protein that stimulates transcription by binding to a sterol regulatory element..Proc. Natl. Acad. Sci. USA. 1993; 90: 11603-11607Crossref PubMed Scopus (500) Google Scholar). Lipogenic enzymes, which are involved in energy storage through synthesis of fatty acids and triglycerides, are coordinately regulated at the transcriptional level during different metabolic states. Recent in vivo studies demonstrated that SREBP-1c plays a crucial role in the dietary regulation of most hepatic lipogenic genes, whereas SREBP-2 is actively involved in the transcription of cholesterogenic enzymes. These include studies of the effects of the absence or over-expression of SREBP-1 on hepatic lipogenic gene expression (16Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M. Goldstein J.L. Overproduction of cholesterol and fatty acids causes massive liver enlargement in transgenic mice expressing truncated SREBP-1a..J. Clin. Invest. 1996; 98: 1575-1584Crossref PubMed Scopus (698) Google Scholar, 17Shimano H. Yahagi N. Amemiya-Kudo M. Hasty A. Osuga J. Tamura Y. Shionoiri F. Iizuka Y. Ohashi K. Harada K. Gotoda T. Ishibashi S. Yamada N. Sterol regulatory element-binding protein-1 as a key transcription factor for nutritional induction of lipogenic enzyme genes..J. Biol. Chem. 1999; 274: 35832-35839Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar, 18Horton J.D. Shimomura I. Brown M.S. Hammer R.E. Goldstein J.L. Shimano H. Activation of cholesterol synthesis in preference to fatty acid synthesis in liver and adipose tissue of transgenic mice overproducing sterol regulatory element-binding protein-2..J. Clin. Invest. 1998; 101: 2331-2339Crossref PubMed Google Scholar), as well as physiological changes of SREBP-1c protein in normal mice after dietary manipulation, such as placement on high carbohydrate diets, PUFA-enriched diets, and fasting-refeeding regimens (19Horton J.D. Bashmakov Y. Shimomura I. Shimano H. Regulation of sterol regulatory element binding proteins in livers of fasted and refed mice..Proc. Natl. Acad. Sci. USA. 1998; 95: 5987-5992Crossref PubMed Scopus (537) Google Scholar, 20Yahagi N. Shimano H. Hasty A. Amemiya-Kudo M. Okazaki H. Tamura Y. Iizuka Y. Shionoiri F. Ohashi K. Osuga J. Harada K. Gotoda T. Nagai R. Ishibashi S. Yamada N. A crucial role of sterol regulatory element-binding protein-1 in the regulation of lipogenic gene expression by polyunsaturated fatty acids..J. Biol. Chem. 1999; 274: 35840-35844Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 21Xu J. Nakamura M.T. Cho H.P. Clarke S.D. Sterol regulatory element binding protein-1 expression is suppressed by dietary polyunsaturated fatty acids. A mechanism for the coordinate suppression of lipogenic genes by polyunsaturated fats..J. Biol. Chem. 1999; 274: 23577-23583Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 22Kim H.J. Takahashi M. Ezaki O. Fish oil feeding decreases mature sterol regulatory element-binding protein 1 (SREBP-1) by down-regulation of SREBP-1c mRNA in mouse liver. A possible mechanism for down-regulation of lipogenic enzyme mRNAs..J. Biol. Chem. 1999; 274: 25892-25898Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). Previous reports on the regulation of SREBP-1c have all demonstrated the induction to be at the mRNA level. Promoter analysis revealed that the expression of the SREBP-1c gene is regulated by two factors: SREBP itself, forming an autoloop, and liver X-activated receptor/retinoic acid receptor (LXR/RXR) oxysterol receptor (23Amemiya-Kudo M. Shimano H. Yoshikawa T. Yahagi N. Hasty A.H. Okazaki H. Tamura Y. Shionoiri F. Iizuka Y. Ohashi K. Osuga J-i. Harada K. Gotoda T. Sato R. Kimura S. Ishibashi S. Yamada N. Promoter analysis of the mouse sterol regulatory element-binding protein (SREBP)-1c gene..J. Biol. Chem. 2000; 275: 31078-31085Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 24Repa J.J. Liang G. Ou J. Bashmakov Y. Lobaccaro J.M. Shimomura I. Shan B. Brown M.S. Goldstein J.L. Mangelsdorf D.J. Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRalpha and LXRbeta..Genes Dev. 2000; 14: 2819-2830Crossref PubMed Scopus (1414) Google Scholar, 25Yoshikawa T. Shimano H. Amemiya-Kudo M. Yahagi N. Hasty A.H. Matsuzaka T. Okazaki H. Tamura Y. Iizuka Y. Ohashi K. Osuga J. Harada K. Gotoda T. Kimura S. Ishibashi S. Yamada N. Identification of liver X receptor-retinoid X receptor as an activator of the sterol regulatory element-binding protein 1c gene promoter..Mol. Cell. Biol. 2001; 21: 2991-3000Crossref PubMed Scopus (434) Google Scholar, 35Schultz J.R. Tu H. Luk A. Repa J.J. Medina J.C. Li L. Schwendner S. Wang S. Thoolen M. Mangelsdorf D.J. Lustig K.D. Shan B. Role of LXRs in control of lipogenesis..Genes Dev. 2000; 14: 2831-2838Crossref PubMed Scopus (1388) Google Scholar). In the screening of SREBP-activated genes, we cloned a mammalian fatty acid elongase, designated fatty acyl-CoA elongase (FACE). Our current data suggest that the enzyme activity of this clone explains the missing identity step in the conversion of C16 to C18 fatty acids. In addition, the nutritional regulation of this SREBP-regulated FACE expression is consistent with its roles as a lipogenic enzyme. We purchased fatty acids from Sigma, restriction enzymes from New England Biolabs, redivue [α-32P]dCTP (6,000 Ci/mmol) from Amersham Pharmacia, and radioactive [2-14C]malonyl-CoA (51 mCi/mmol) from New England Nuclear. Standard molecular biology techniques were used. DNA sequencing was performed with the CEQ™ dye terminator cycle sequencing kit and CEQ2000 DNA Analysis System (Beckman Coulter). An expression cDNA library of SREBP-1a transgenic liver (16Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M. Goldstein J.L. Overproduction of cholesterol and fatty acids causes massive liver enlargement in transgenic mice expressing truncated SREBP-1a..J. Clin. Invest. 1996; 98: 1575-1584Crossref PubMed Scopus (698) Google Scholar) was prepared as previously described for construction of a cDNA library of SREBP-1 deficient mouse adipose tissue, except that poly(A)+ RNA was prepared from livers of SREBP-1a transgenic mice (25Yoshikawa T. Shimano H. Amemiya-Kudo M. Yahagi N. Hasty A.H. Matsuzaka T. Okazaki H. Tamura Y. Iizuka Y. Ohashi K. Osuga J. Harada K. Gotoda T. Kimura S. Ishibashi S. Yamada N. Identification of liver X receptor-retinoid X receptor as an activator of the sterol regulatory element-binding protein 1c gene promoter..Mol. Cell. Biol. 2001; 21: 2991-3000Crossref PubMed Scopus (434) Google Scholar). From a DNA microarray system using hepatic poly(A)+ RNA of SREBP-1a transgenic and non-transgenic littermate mice (GenomeIncyte), we identified an EST clone (GenBank ID number AA239254) that was activated 19.5-fold in SREBP-1a transgenic liver as compared with wild-type liver. Using this sequence information, a [α-32P]dCTP labeled DNA probe was prepared and used in the screening of an SREBP-1a transgenic mouse liver cDNA library by colony hybridization. Positive clones were sequenced; however, the clones were the 3′-fragment of the cDNA. To isolate the 5′ ends of the clones, the 5′-RACE method was used. Poly(A)+ RNA was isolated using oligo-dT Latex (TaKaRa) from the liver of a SREBP-1a transgenic mouse and was used for cDNA synthesis and amplification with the 5′-Full RACE Core Set (TaKaRa). The cDNA sequence was subjected to BLAST search of mouse EST database and UniGene mouse database. The search revealed a cluster of mouse EST sequences (UniGene cluster ID number Mm. 26171) which contained a single open reading frame of 822 bp with similarity to Cig30, another known mouse fatty acid elongase. The putative elongase gene was tentatively designated FACE. Primers TMBSP1 (5′-TGG ATG CGG ACG CTG GGA GG–3′) and TMBAP1 (5′-AGT TGC ACT CAG CGA GTC CT-3′) were designed based on the putative FACE sequence and used to amplify the full length FACE cDNA from SREBP-1a transgenic mouse liver cDNA. The 1.1 kb PCR amplified product was subcloned into pGEM-T easy vector (Promega) and sequenced. The FACE cording sequence was isolated from this plasmid by NotI digestion and blunt ended using DNA Blunting Kit (TaKaRa), and inserted into SmaI site of expression CMV7 vector. HEK-293 cells were grown at 37°C in an atmosphere of 5% CO2 in DMEM containing 25 mM glucose, 100 U/ml penicillin, and 100 μg/ml streptomycin sulfate supplemented with 10% FBS on 100 mm culture plates. At 80% confluency, the mouse FACE expression plasmid or the empty plasmid CMV7 (10 μg) was transfected into cells using SuperFect Transfection Reagent (Qiagen) according to the manufacturer's protocol. After transfection, cells were incubated with DMEM plus 10% FBS for 24 h. The microsomal fractions from the cells were prepared as previously described, with some modification (26Roberts S.B. Ripellino J.A. Ingalls K.M. Robakis N.K. Felsenstein K.M. Non-amyloidogenic cleavage of the beta-amyloid precursor protein by an integral membrane metalloendopeptidase..J. Biol. Chem. 1994; 269: 3111-3116Abstract Full Text PDF PubMed Google Scholar). Twenty four hours after the transfection, cells were washed with PBS and scraped in 5 ml of ice-cold 0.25 M sucrose, 0.02 M HEPES, pH7.5. The cells were washed and resuspended in 3 ml of ice-cold sucrose/HEPES and dounce-homogenized. The homogenate was centrifuged 1,000 g for 7 min at 4°C. The pellet was resuspended in 1 ml of sucrose/HEPES, dounce-homogenized, and the suspension was centrifuged at 1,000 g for 7 min at 4°C. The supernatants were combined and re-centrifuged at 2,000 g for 30 min at 4°C. Supernatant from this centrifugation was centrifuged at 105,000 g for 60 min at 4°C. The resultant pellets were resuspended in 100 μl of 0.1 M Tris-HCl, pH 7.4 and used for fatty acid elongation assay. Microsomal fatty acid elongation activity was assayed by the measurement of [2-14C]malonyl-CoA incorporation into exogenous acyl-CoAs as described previously (27Alegret M. Cerqueda E. Ferrando R. Vazquez M. Sanchez R.M. Adzet T. Merlos M. Laguna J.C. Selective modification of rat hepatic microsomal fatty acid chain elongation and desaturation by fibrates: relationship with peroxisome proliferation..Br. J. Pharmacol. 1995; 114: 1351-1358Crossref PubMed Scopus (33) Google Scholar), with some modification. The assay mixtures (0.25 ml total, including protein addition) contained 100 μM Tris-HCl, pH 7.4, 60 μM palmitoyl-CoA, 500 μM NADPH, and 30 μg of freshly obtained microsomal protein. After 2 min of preincubation at 37°C, the reaction was initiated by the addition of 60 μM malonyl-CoA (containing 0.037 μCi of [2-14C]malonyl-CoA) and carried out for 5 min at 37°C. The incubation was terminated by addition of 0.5 ml of 15% KOH in methanol and saponified at 65°C for 45 min. Then the samples were cooled and acidified with 0.5 ml of ice-cold 5 N HCl. Free fatty acids were extracted from the mixture three times with 1 ml of hexane (total vol 3 ml). The pooled hexane fractions were dried under vacuum, and after addition of 3 ml of scintillation mixture, the radioactivity incorporated was counted (BECKMAN LS6500). Blanks were carried out in parallel reactions incubated without microsomal fractions. All mice were housed in a controlled environment with a 12-h light/dark cycle and free access to water and diet. For fatty acid or drug experiments, 7-week-old male C57BL/6J mice (21–23 g) were purchased from CLEA (Tokyo, Japan) and adapted to the environment for 1 week. Prior to sacrifice, each group of animals was fed a diet containing the indicated fatty acids and drugs for 7 days. SREBP-1a, and -1c, and -2 transgenic mice (16Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M. Goldstein J.L. Overproduction of cholesterol and fatty acids causes massive liver enlargement in transgenic mice expressing truncated SREBP-1a..J. Clin. Invest. 1996; 98: 1575-1584Crossref PubMed Scopus (698) Google Scholar, 18Horton J.D. Shimomura I. Brown M.S. Hammer R.E. Goldstein J.L. Shimano H. Activation of cholesterol synthesis in preference to fatty acid synthesis in liver and adipose tissue of transgenic mice overproducing sterol regulatory element-binding protein-2..J. Clin. Invest. 1998; 101: 2331-2339Crossref PubMed Google Scholar, 28Shimano H. Horton J.D. Shimomura I. Hammer R.E. Brown M.S. Goldstein J.L. Isoform 1c of sterol regulatory element binding protein is less active than isoform 1a in livers of transgenic mice and in cultured cells..J. Clin. Invest. 1997; 99: 846-854Crossref PubMed Scopus (683) Google Scholar) and wild-type controls (non-transgenic littermates of SREBP-1a transgenic mice) were put on a high protein/low carbohydrate diet for 2 weeks to induce the transgene expression, and were fasted for 12 h prior sacrifice. For fasting and refeeding treatment, SREBP-1 deficient (29Shimano H. Shimomura I. Hammer R.E. Herz J. Goldstein J.L. Brown M.S. Horton J.D. Elevated levels of SREBP-2 and cholesterol synthesis in livers of mice homozygous for a targeted disruption of the SREBP-1 gene..J. Clin. Invest. 1997; 100: 2115-2124Crossref PubMed Scopus (353) Google Scholar) and wild-type mice were fasted for 24 h and fed a high sucrose/fat free diet for 12 h. Ob/+ mice on a C57Bl/6 background were purchased from Jackson Laboratories. Ob/+ mice were crossed to obtain leptin deficient ob/ob mice and wild-type mice. At 12 weeks old, ob/ob and wild-type mice were sacrificed in the early light phase following a 2 h fast. Total RNA was extracted from mouse livers, white adipose tissue (WAT), and various tissues using TRIZOL Reagent (Life Technologies, Inc.). RNA samples were run on a 1% agarose gel containing formaldehyde and transferred to a nylon membrane (Hybond-N, Amersham Pharmacia Biotech). The probes used were labeled with [α-32P]dCTP using the Megaprime DNA Labeling System kit (Amersham Pharmacia Biotech). The cDNA probe for mouse FACE was prepared by digesting the cloned cDNA with NotI. The cDNA probes for mouse SREBP-1 and ribosomal phosphoprotein PO (36B4) were prepared as described previously (17Shimano H. Yahagi N. Amemiya-Kudo M. Hasty A. Osuga J. Tamura Y. Shionoiri F. Iizuka Y. Ohashi K. Harada K. Gotoda T. Ishibashi S. Yamada N. Sterol regulatory element-binding protein-1 as a key transcription factor for nutritional induction of lipogenic enzyme genes..J. Biol. Chem. 1999; 274: 35832-35839Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar). The membranes were hybridized with the radiolabeled probe in Rapid-hyb Buffer (Amersham Pharmacia Biotech) at 65°C and washed in 0.1 × SSC, 0.1% SDS at 65°C. The resulting bands were quantified by exposure of the filters to BAS2000 with BAS station software (Fuji Photo Film Co., Ltd). DNA microarray analysis identified an EST clone whose expression was increased 19.5-fold in the livers of SREBP-1a transgenic mice as compared with wild-type mice. Using this EST clone (GenBank ID number AA239254) as a probe, we obtained the full-length 6 kb cDNA by screening an SREBP-1a transgenic liver cDNA library, followed by 5′ RACE. As shown in Fig. 1A, nucleotide sequence of this clone revealed that it encodes a putative protein of 267 amino acid residues with a theoretical molecular mass of 31.6 kDa and very basic pI of 9.38. We found a human homolog (GenBank ID number AK027031) of mouse FACE after searching BLAST database. The predicted amino acid sequence revealed that 96% identical and 97% similarity in mouse and human homologs (Fig. 1B). Hydropathy analyses by the Kyte-Doolittle algorithm (30Kyte J. Doolittle R.F. A simple method for displaying the hydropathic character of a protein..J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (17170) Google Scholar) suggest that this predicted protein contains five transmembrane regions, typical for members of the elongase family (Fig. 1C). An HXXHH motif, often present in desaturase/hydroxylase enzymes containing di-iron-oxo cluster (Fe-O-Fe) proteins (31Shanklin J. Whittle E. Fox B.G. Eight histidine residues are catalytically essential in a membrane-associated iron enzyme, stearoyl-CoA desaturase, and are conserved in alkane hydroxylase and xylene monooxygenase..Biochemistry. 1998; 33: 12787-12794Crossref Scopus (647) Google Scholar), was found between predicted transmembrane regions II and III, at amino acid positions 141–145, and could function to receive electrons from either cytochrome b5 or a cytochrome b5-like domain in an NAD(P)H-dependent way (Fig. 1B, C). The COOH terminus of the FACE polypeptide contains a lysine residue in position −3 (KKXX-like motif), suggesting that the predicted protein is located in the endoplasmic reticulum (ER) membrane (Fig. 1B) (32Jackson M.R. Nilsson T. Peterson P.A. Identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum..EMBO J. 1990; 9: 3153-3162Crossref PubMed Scopus (724) Google Scholar). The predicted amino acid sequence of mouse FACE showed a considerable similarity to Cig30 (44% identical), Ssc1 (29%), Ssc2 (26%), and Elovl4 (26%), all of which are known to be involved in the elongation of fatty acids by two carbon atoms, and they all contain 100% conserved HXXHH motif characteristic of this protein family (Fig. 2).Fig. 2.Amino acid sequence alignment of mouse FACE homologs. Amino acid positions conserved in the homologs are indicated under the protein sequence. The HXXHH motif, characteristic of desaturase/hydroxylase enzymes containing a diiron-oxo cluster (Fe-O-Fe) is underlined.View Large Image Figure ViewerDownload Hi-res image Download (PPT) This cDNA clone is highly likely to encode a mammalian fatty acid elongase, and is tentatively designated FACE. When the FACE cDNA was over-expressed in HEK293 cells supplemented with various fatty acids, gas chromatography analysis of the cellular fatty acids showed a trend that over-expression of FACE caused a slight increase in the relative amounts of stearic acid (C18:0) and oleic acid (C18:1n-9), accompanying a decrease in palmitic acid (C16:0) and myristic acid (C14:0) in the cells as compared with mock transfected cells (CMV7) (data not shown). These data suggested that the cells transfected with FACE were capable of synthesizing C18:0 from C16:0, indicating that FACE is involved in the elongation of C16:0 and that FACE may have an elongase acitivity for C14:0. To verify that FACE plays a role in the conversion of C16:0 to C18:0, we performed in vitro microsomal fatty acid elongation assays. Microsomal fatty acid elongation activity was assayed by the measurement of [2-14C]malonyl-CoA incorporation into exogenously added acyl-CoA esters of saturated and mono-unsaturated fatty acids with chain length 12 to 18, namely lauroyl-CoA (C12:0), myristoyl-CoA (C14:0), palmitoyl-CoA (C16:0), palmitoleoyl-CoA (C16:1n-7), stearoyl-CoA (C18:0), and oleoyl-CoA (C18:1n-9). Compared with control cells that had been transfected with empty plasmid CMV7, the transfection of the FACE
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