Elovl2 ablation demonstrates that systemic DHA is endogenously produced and is essential for lipid homeostasis in mice
2014; Elsevier BV; Volume: 55; Issue: 4 Linguagem: Inglês
10.1194/jlr.m046151
ISSN1539-7262
AutoresAnna M. Pauter, Petter Olsson, Abolfazl Asadi, Bengt G. Herslöf, Robert I. Csikasz, Damir Zadravec, Anders Jacobsson,
Tópico(s)Fatty Acid Research and Health
ResumoThe potential role of endogenously synthesized PUFAs is a highly overlooked area. Elongation of very long-chain fatty acids (ELOVLs) in mammals is catalyzed by the ELOVL enzymes to which the PUFA elongase ELOVL2 belongs. To determine its in vivo function, we have investigated how ablation of ELOVL2, which is highly expressed in liver, affects hepatic lipid composition and function in mice. The Elovl2−/− mice displayed substantially decreased levels of 22:6(n-3), DHA, and 22:5(n-6), docosapentaenoic acid (DPA) n-6, and an accumulation of 22:5(n-3) and 22:4(n-6) in both liver and serum, showing that ELOVL2 primarily controls the elongation process of PUFAs with 22 carbons to produce 24-carbon precursors for DHA and DPAn-6 formation in vivo. The impaired PUFA levels positively influenced hepatic levels of the key lipogenic transcriptional regulator sterol-regulatory element binding protein 1c (SREBP-1c), as well as its downstream target genes. Surprisingly, the Elovl2−/− mice were resistant to hepatic steatosis and diet-induced weight gain, implying that hepatic DHA synthesis via ELOVL2, in addition to controlling de novo lipogenesis, also regulates lipid storage and fat mass expansion in an SREBP-1c-independent fashion. The changes in fatty acid metabolism were reversed by dietary supplementation with DHA. The potential role of endogenously synthesized PUFAs is a highly overlooked area. Elongation of very long-chain fatty acids (ELOVLs) in mammals is catalyzed by the ELOVL enzymes to which the PUFA elongase ELOVL2 belongs. To determine its in vivo function, we have investigated how ablation of ELOVL2, which is highly expressed in liver, affects hepatic lipid composition and function in mice. The Elovl2−/− mice displayed substantially decreased levels of 22:6(n-3), DHA, and 22:5(n-6), docosapentaenoic acid (DPA) n-6, and an accumulation of 22:5(n-3) and 22:4(n-6) in both liver and serum, showing that ELOVL2 primarily controls the elongation process of PUFAs with 22 carbons to produce 24-carbon precursors for DHA and DPAn-6 formation in vivo. The impaired PUFA levels positively influenced hepatic levels of the key lipogenic transcriptional regulator sterol-regulatory element binding protein 1c (SREBP-1c), as well as its downstream target genes. Surprisingly, the Elovl2−/− mice were resistant to hepatic steatosis and diet-induced weight gain, implying that hepatic DHA synthesis via ELOVL2, in addition to controlling de novo lipogenesis, also regulates lipid storage and fat mass expansion in an SREBP-1c-independent fashion. The changes in fatty acid metabolism were reversed by dietary supplementation with DHA. As a source of energy and as structural components of membranes, omega-3 (n-3) and omega-6 (n-6) PUFAs have been shown to have significant roles in many biological functions, including immunity, growth, and metabolism in mammals. Dietary PUFAs, and especially DHA, have been shown to influence hepatic liver metabolism and prevent the development of liver steatosis (1Jump D.B. Clarke S.D. Regulation of gene expression by dietary fat.Annu. Rev. Nutr. 1999; 19: 63-90Crossref PubMed Scopus (546) Google Scholar, 2Sekiya M. Yahagi N. Matsuzaka T. Najima Y. Nakakuki M. Nagai R. Ishibashi S. Osuga J. Yamada N. Shimano H. Polyunsaturated fatty acids ameliorate hepatic steatosis in obese mice by SREBP-1 suppression.Hepatology. 2003; 38: 1529-1539Crossref PubMed Scopus (336) Google Scholar, 3González-Périz A. Horrillo R. Ferré N. Gronert K. Dong B. Morán-Salvador E. Titos E. Martínez-Clemente M. López-Parra M. 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Rev. 1973; 31: 248-249PubMed Google Scholar). The biosynthesis of PUFAs of the n-6 and n-3 series occurs via sequential desaturation and elongation steps by the Δ5- and Δ6-fatty acid desaturases (FADSs) 1 and 2, and the fatty acid elongation of very long-chain fatty acids (ELOVLs) 5, 2, and 4 (6Guillou H. Zadravec D. Martin P.G. Jacobsson A. The key roles of elongases and desaturases in mammalian fatty acid metabolism: insights from transgenic mice.Prog. Lipid Res. 2010; 49: 186-199Crossref PubMed Scopus (571) Google Scholar). However, the biosynthesis of the omega-6 PUFA C22:5n-6, docosapentaenoic acid (DPA) n-6, and the omega-3 PUFA C22:6n-3, DHA, requires 24-carbon intermediates, which are subsequently desaturated prior to chain shortening through partial β-oxidation, ultimately yielding DPA and DHA (7Sprecher H. Metabolism of highly unsaturated n-3 and n-6 fatty acids.Biochim. Biophys. Acta. 2000; 1486: 219-231Crossref PubMed Scopus (654) Google Scholar, 8Voss A. Reinhart M. Sankarappa S. Sprecher H. The metabolism of 7,10,13,16,19-docosapentaenoic acid to 4,7,10,13,16,19-docosahexaenoic acid in rat liver is independent of a 4-desaturase.J. Biol. Chem. 1991; 266: 19995-20000Abstract Full Text PDF PubMed Google Scholar). ELOVL5 has been proposed to be involved in the elongation of 18- and 20-carbon PUFA substrates, while data indicate that ELOVL2 and ELOVL4 elongate C20–C24 and C26–C34 PUFAs, respectively (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, 10Moon Y.A. Shah N.A. Mohapatra S. Warrington J.A. Horton J.D. Identification of a mammalian long chain fatty acyl elongase regulated by sterol regulatory element-binding proteins.J. Biol. Chem. 2001; 276: 45358-45366Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 11Wang Y. Torres-Gonzalez M. Tripathy S. Botolin D. Christian B. Jump D.B. Elevated hepatic fatty acid elongase-5 activity affects multiple pathways controlling hepatic lipid and carbohydrate composition.J. Lipid Res. 2008; 49: 1538-1552Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 12Gregory M.K. Cleland L.G. James M.J. Molecular basis for differential elongation of omega-3 docosapentaenoic acid by the rat Elovl5 and Elovl2.J. Lipid Res . 2013; 54: 2851-2857Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). While ELOVL5, FADS1, and FADS2 are expressed to a certain extent in all tissues tested, the level of ELOVL2 is significant in liver, testis, uterus, placenta, mammary gland, retina, and certain areas of the brain, all of which are tissues that are documented as being rich in DHA (13Tvrdik 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: 707-718Crossref PubMed Scopus (182) Google Scholar, 14Ohno Y. Suto S. Yamanaka M. Mizutani Y. Mitsutake S. Igarashi Y. Sassa T. Kihara A. ELOVL1 production of C24 acyl-CoAs is linked to C24 sphingolipid synthesis.Proc. Natl. Acad. Sci. USA. 2010; 107: 18439-18444Crossref PubMed Scopus (240) Google Scholar). In contrast to testis and retina, PUFAs longer than C24 are almost undetectable in liver, mainly due to the low level of Elovl4 expression in this tissue, while PUFAs up to C22 are quite abundant within both the phospholipid and TG pools of hepatocytes (15Tikhonenko M. Lydic T.A. Wang Y. Chen W. Opreanu M. Sochacki A. McSorley K.M. Renis R.L. Kern T. Jump D.B. et al.Remodeling of retinal fatty acids in an animal model of diabetes: a decrease in long chain polyunsaturated fatty acids is associated with a decrease in fatty acid elongases Elovl2 and Elovl4.Diabetes. 2010; 59: 219-227Crossref PubMed Scopus (100) Google Scholar). As the first enzyme in the PUFA elongation chain, ELOVL5 activity is suggested to affect multiple pathways regulating hepatic lipid and carbohydrate composition in mammals (11Wang Y. Torres-Gonzalez M. Tripathy S. Botolin D. Christian B. Jump D.B. Elevated hepatic fatty acid elongase-5 activity affects multiple pathways controlling hepatic lipid and carbohydrate composition.J. Lipid Res. 2008; 49: 1538-1552Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), while the role for ELOVL2 in liver is unknown. Our previous study, concerning the role of ELOVL2 in sperm maturation and male fertility, revealed that Elovl2−/− mice are infertile and heterozygote Elovl2+/− C57Bl/6 mice exhibit haploinsufficiency that gives rise to impaired spermatides in >99% of all male mice, which hindered us from obtaining homozygous Elovl2−/− mice (16Zadravec D. Tvrdik P. Guillou H. Haslam R. Kobayashi T. Napier J.A. Capecchi M.R. Jacobsson A. ELOVL2 controls the level of n-6 28:5 and 30:5 fatty acids in testis, a prerequisite for male fertility and sperm maturation in mice.J. Lipid Res. 2011; 52: 245-255Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). To overcome this, we backcrossed Elovl2+/− females with 129S2/Sv males enabling the production of fertile Elovl2+/− males and subsequently generation of Elovl2−/− mice. In vitro data have suggested a role of ELOVL2 in the elongation of C20–C24 PUFAs. By using ELOVL2-ablated mice, we now show that the major in vivo substrates of ELOVL2 are C22:5n-3 and C22:4n-6. As a consequence, ELOVL2 completes the final elongation step in the synthesis of C22:5n-6 and C22:6n-3 in liver. Our data also reveal, for the first time, the physiological consequences of impaired ELOVL2 activity and endogenous DHA synthesis. The Elovl2-ablated mice have significantly distorted levels of C22 PUFAs in serum that correlate extremely well with the levels seen in both the phospholipid and TG pools in liver. Particularly, the mutant mice show a massive (20-fold) reduction in the ratio of the omega-3 PUFAs 22:6n-3 and 22:5n-3, implying hepatic ELOVL2 as a dominating factor in the control of serum levels of DHA. As a consequence, despite an induced nuclear form of hepatic sterol-regulatory element binding protein 1c (SREBP-1c), the Elovl2−/− mice show a reduced capacity to accumulate fat, which is reversible by dietary DHA supplementation. Elovl2−/− mice were generated as described previously (16Zadravec D. Tvrdik P. Guillou H. Haslam R. Kobayashi T. Napier J.A. Capecchi M.R. Jacobsson A. ELOVL2 controls the level of n-6 28:5 and 30:5 fatty acids in testis, a prerequisite for male fertility and sperm maturation in mice.J. Lipid Res. 2011; 52: 245-255Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) and backcrossed 129S2/Sv for four generations. All animals were housed at room temperature and maintained on a 12 h light/dark cycle. Ten- to 17-week-old male mice were single caged and fed standard chow diet for 12 weeks (<10% kcal fat, Labfor R70; Lantmännen, Sweden) or a high-fat diet (45% kcal fat, D12451; Research Diets, New Brunswick, NJ). In addition, 17- to 18-week-old male mice were fed standard chow diet or DHA-enriched chow diet for 2 weeks (Quimper, France) followed by 2 weeks of high-fat diet treatment. For the fatty acid composition of diets, see supplementary Table II. All animals were fed ad libitum and had free access to water. As control mice, age-matched littermates from a heterozygote breeding were used and housed under the same conditions as the Elovl2−/− mice. Body weight and food consumption were measured weekly as indicated in the Results section. At the end of the study, animals were euthanized with CO2. Relevant tissues were frozen in liquid nitrogen and stored at −80°C until required. All studies were carried out with ethical permission from the Animal Ethics Committee of the North Stockholm region, Sweden. Liver extracts were prepared by grinding a small piece of frozen sample to a fine powder using a ceramic mortar in the presence of liquid nitrogen. Fifty milligrams was transferred to a glass test tube with a teflon-lined screw cap. Twenty microliters of triheptadecanoin (10.0 mg/ml in chloroform) and 40 µl of 1,2-diheptadecanoyl-sn-glycero-3-phosphatidylcholine (5.0 mg/ml in chloroform) were added, and the sample was dried in a stream of nitrogen. The extraction protocol then followed the procedure earlier published by Matyash et al. (17Matyash V. Liebisch G. Kurzchalia T.V. Shevchenko A. Schwudke D. Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics.J. Lipid Res. 2008; 49: 1137-1146Abstract Full Text Full Text PDF PubMed Scopus (1380) Google Scholar). Prior to HPLC separation, the samples were redissolved in chloroform-methanol (1:2, v/v). Serum extracts were obtained by centrifugation of fresh blood samples at 1,800 g for 20 min according to Matthan et al. (18Matthan N.R. Ip B. Resteghini N. Ausman L.M. Lichtenstein A.H. Long-term fatty acid stability in human serum cholesteryl ester, triglyceride, and phospholipid fractions.J. Lipid Res. 2010; 51: 2826-2832Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) and were then stored in −80°C until use. Fifty microliters was transferred to a glass test tube with a teflon-lined screw cap. Twenty microliters of triheptadecanoin (10.0 mg/ml in chloroform) was added, the sample was dried in a stream of nitrogen, and lipids were extracted according to Matyash et al. (17Matyash V. Liebisch G. Kurzchalia T.V. Shevchenko A. Schwudke D. Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics.J. Lipid Res. 2008; 49: 1137-1146Abstract Full Text Full Text PDF PubMed Scopus (1380) Google Scholar). All solvents were of HPLC grade and purchased from Rathburn Chemicals Ltd. (Walkerburn, Scotland). Lipid standards were purchased from Larodan Fine Chemicals AB (Malmö, Sweden) with purity ≥99%. Boron trifluoride (BF3, 14% in methanol) was purchased from Sigma Aldrich (Milwaukee, WI). Fractionation of liver samples was performed using preparative HPLC by modification of an earlier published method (19Olsson P. Holmback J. Herslof B. Separation of lipid classes by HPLC on a cyanopropyl column.Lipids. 2012; 47: 93-99Crossref PubMed Scopus (23) Google Scholar). An HP-1050 quaternary pump (Agilent Technologies Inc., Santa Clara, CA) was fitted to a Reprosil-Pur 120 CN column [250 mm × 10 mm inner diameter (ID), 5 µm particle size; Dr. Maisch GmbH, Ammerbuch, Germany] and a Reprosil-Pur CN guard column (30 mm × 10 mm ID, 5 µm particle size; Dr. Maisch GmbH). An evaporative light-scattering detector (DDL21; Cunow, St. Christophe, France) was used for detection. Elution was performed using a binary mixture consisting of mobile phase A (heptane) and mobile phase B [toluene-methanol-acetic acid-triethylamine (60:40:0.2:0.1, by weight)]. A gradient was applied after 3 min by increasing B from 10% to 40% in 7 min. Then B was further increased to 50% before being decreased again to 10% in 1 min. The total mobile flow rate was set to 4.7 ml/min and split 1:10 using a t-connection, where the lower-flow end was connected to the detector and the high-flow end was used for fraction collection. Eighty microliters of mouse liver extract was injected, and the neutral lipid fraction was collected from 2 to 5 min, and the polar lipid fraction from 11 to 14 min into glass test tubes provided with teflon-lined screw caps. After collection, all samples were dried in a stream of nitrogen and then subjected to transesterification and analyzed by GC as described previously (20Idborg H. Olsson P. Leclerc P. Raouf J. Jakobsson P-J. Korotkova M. Effects of mPGES-1 deletion on eicosanoid and fatty acid profiles in mice.Prostaglandins Other Lipid Mediat. 2013; 107: 18-25Crossref PubMed Scopus (29) Google Scholar). In vivo MRI using EchoMRI-100TM (Echo Medical Systems, Houston, TX) was performed in order to measure body fat and lean content. To determine resting metabolic rate, oxygen consumption was measured by indirect calorimetry (INCA systems; Somedic, Hörby, Sweden) as previously described (21Alberts P. Johansson B.G. McArthur R.A. Characterization of energy expenditure in rodents by indirect calorimetry.Curr. Protoc. Neurosci. 2006; 36: 9.23D.1-9.23D.17Crossref Scopus (17) Google Scholar). Mice were subjected to oxygen consumption measurements at room temperature for 22 h, starting at 0800. Animals were fed ad libitum and had free access to water. Animals in their home cages were put into sealed chambers (vol 4 liters). The air leaving the chamber was dried with silica gel (Safegel 1–3 mm with yellow moisture indicator, Merck Prolabo; VMR International, West Chester, PA). The zirconium oxide sensors were calibrated daily using two reference gases containing 18% and 25% oxygen in nitrogen before measurement. Measurements proceeded under a constant airflow rate (1liter/min). Oxygen consumption (VO2) and carbon dioxide production (VCO2) were recorded every 2 min. Resting energy expenditure (REE) was calculated according to the Weir formula (REE = 16.3 VO2 + 4.57 VCO2) and normalized to the lean weight per animal. Livers were fixed in 10% neutral buffered formalin, sectioned, stained with hematoxylin and eosin, and examined by light microscopy histological evaluation. RNA was isolated with TRI Reagent (Sigma Aldrich), and total RNA was isolated following the manufacturer's procedure. For real-time PCR, 500 ng total RNA was reverse transcribed using random hexamer primers, deoxynucleoside triphosphates, MultiScribe reverse transcriptase, and RNase inhibitor (Applied Biosystems, Foster City, CA). cDNA samples were diluted 1:10, and aliquots of 2 μl of the sample cDNA were mixed with SYBR Green JumpStart Taq ReadyMix (Sigma Aldrich), prevalidated primers, and diethylpyrocarbonate-treated water and were measured in duplicate for each sample. Primers used were fatty acid synthase (FAS), SREBP-1c, sterol-CoA desaturase 1 (SCD1), PPARγ, and phosphoenolpyruvate carboxykinase 1 (Pck1). For sequences, see supplementary Table VIII. Expression analysis was performed using the BioRad detection system. Data were normalized to the housekeeping gene 18S. Five hundred milligrams of frozen liver was placed in ice-cold PBS and subsequently homogenized in 10 mM HEPES, pH 7.6, 25 mM KCl, 0.15 mM spermine, 0.5 mM spermidine, 1 mM EDTA, 2 mM sucrose, 10% glycerol, 2 mM DTT, 0.1 mM PMSF, and one protease inhibitor tablet (Complete Protease Inhibitor tablets; Roche) per 10 ml and subjected to centrifugation at 20,000 rpm for 1 h at 4°C in a JA20 rotor. The pellet was resuspended in 10 mM HEPES, pH 7.6, 100 mM KCl, 0.1 mM EDTA, 3 mM MgCl2, 10% glycerol, 1 mM DTT, 0.1 mM PMSF, and one protease inhibitor tablet (Complete Protease Inhibitor tablets; Roche) per 10 ml, and 1/10 vol of 4 M (NH4)2SO4 was added. After 40 min of incubation on ice, the solution was subjected to ultracentrifugation at 85,000 rpm for 45 min at 4°C in a TLV 100K rotor. The supernatant was collected as a nuclear protein extract. Protein concentration was determined using the Lowry Assay. For immunoblotting of SREBP-1c, the nuclear protein extracts from each sample (10 μg/lane) were separated by 10% SDS-PAGE and blotted to a polyvinylidene difluoride transfer membrane (Amersham Hybond-P; GE Healthcare) in a semidry system. The membrane was incubated for 1 h in 5% fat-free milk, then overnight with the primary SREBP-1c antibody (2A4: sc-13551; Santa Cruz Biotechnology) diluted 1:1,000 in 5% BSA, 1× TBS, and Tween-20 and for 1 h with horseradish peroxidase-conjugated secondary antibody (anti-mouse; Cell Signaling) diluted 1:2,000 in 5% fat-free milk, 10× TBS, and Tween-20. The membrane was washed with TBS and Tween-20 2 × 5 min and 15 min after each incubation time. Proteins were visualized using an ECL Plus kit (Amersham Bioscience) and detected in an LAS-1000 CCD camera (Fuji). Statistical analysis was performed using GraphPad PRISM (San Diego, CA), and statistical differences were calculated with Student's unpaired t-test. Considering the role of ELOVL2 and PUFA synthesis in liver, we analyzed by GC in combination with HPLC fractionation the hepatic fatty acid composition in both the phospholipids and TG pools in Elovl2-ablated mice and compared this with the fatty acid composition in serum. We observed an accumulation of the omega-6 fatty acids 20:4n-6 (arachidonic acid, AA) and 22:4n-6, as well as the omega-3 fatty acids 20:5 (EPA) and 22:5n-3, in both liver and serum of Elovl KO mice compared with wild-type littermates. This was accompanied by a massive (90%) decrease in the levels of 22:5n-6 and 22:6n-3, respectively (Fig. 1A–C). This clearly demonstrates a unique role of ELOVL2 in the elongation process of C22 into C24 PUFAs and, consequently, in the formation of DHA in liver (Fig. 1D). Moreover, the fatty acid profile in serum was reflected entirely by the hepatic lipid composition, indicating liver as the major contributor of serum DHA under standard dietary conditions. It has been reported that one of the beneficial effects of PUFAs, especially DHA, on lipid metabolism is via their involvement in the regulation of the key lipogenic transcriptional factor SREBP-1c. As dietary supplementation with DHA has been shown to both repress hepatic transcription and to positively influence the degradation of the nuclear, active form of SREBP-1c (nSREBP-1c), DHA potentially drives deactivation of SREBP-1c target genes, such as FAS and SCD1, and suppress de novo lipogenesis in mice (22Lin J. Yang R. Tarr P.T. Wu P.H. Handschin C. Li S. Yang W. Pei L. Uldry M. Tontonoz P. et al.Hyperlipidemic effects of dietary saturated fats mediated through PGC-1beta coactivation of SREBP.Cell. 2005; 120: 261-273Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, 23Xu J. Teran-Garcia M. Park J.H. Nakamura M.T. Clarke S.D. Polyunsaturated fatty acids suppress hepatic sterol regulatory element-binding protein-1 expression by accelerating transcript decay.J. Biol. Chem. 2001; 276: 9800-9807Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 24Kim 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). In line with this, the Elovl2-ablated mice, which are deficient in DHA, showed significantly elevated hepatic mRNA levels of SREBP-1c (Fig. 2A), as well as nSREBP-1c protein (Fig. 2B). As expected, upregulation of SREBP-1c resulted in the activation of the downstream target genes for FAS and SCD1 (Fig. 2A). No differences were detected in the level of diacylglycerol acyltransferase 2, carnitine palmitoyltransferase 1, and acetyl-CoA carboxylase, indicating normal TG formation and fatty acid oxidation in the KO mice. These findings are in accordance with previous data showing a negative regulation of SREBP-1c by dietary DHA exposure, as well as with the study on Elovl5-ablated mice that reports an activation of SREBP-1c under conditions of reduced levels of endogenously synthesized PUFAs longer than C18 (25Moon Y.A. Hammer R.E. Horton J.D. Deletion of ELOVL5 leads to fatty liver through activation of SREBP-1c in mice.J. Lipid Res. 2009; 50: 412-423Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Activation of SREBP-1c and its target genes has been proposed as a major factor in the development of hepatic steatosis due to stimulation of fatty acid and TG synthesis (26Horton J.D. Goldstein J.L. Brown M.S. SREBPs: transcriptional mediators of lipid homeostasis.Cold Spring Harb. Symp. Quant. Biol. 2002; 67: 491-498Crossref PubMed Scopus (157) Google Scholar). Recently, Moon and coworkers (25Moon Y.A. Hammer R.E. Horton J.D. Deletion of ELOVL5 leads to fatty liver through activation of SREBP-1c in mice.J. Lipid Res. 2009; 50: 412-423Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) showed that Elovl5 ablation and reduced levels of PUFA lead to steatosis by impaired suppression of SREBP-1c. Surprisingly, despite upregulation in hepatic lipogenic gene expression (Fig. 2A), Elovl2−/− mice did not show accumulation of liver TGs (Fig. 2D), and from the histological analysis, we can conclude that Elovl2−/− liver does not significantly differ from wild-type hepatic tissue and did not show any signs of exaggerated fat storage and steatosis (Fig. 2C). Furthermore, there was no significant difference in body weight, lean weight, fat mass, and energy intake between Elovl2−/− mice and wild-type littermates (supplementary Table I). In addition, we did not observe any disparity regarding fasting blood glucose levels (7.18 and 7.68 mM between wild-type and Elovl2−/− mice, respectively), TG and cholesterol levels, or response to glucose tolerance test between Elovl2−/− mice and wild-type littermates. From indirect calorimetric measurements, we could detect a tendency for the Elovl2-ablated animals to have higher energy expenditure (resting metabolic rate) than wild-type mice in the light period and at the beginning of the dark period (Fig. 2E), although this difference in energy expenditure was slightly reversed during the later stage of the dark phase. When we analyzed the calorimetric parameter RQ, we found that it was significantly lower during the dark period, when the mice are more active and have their major food intake, implying that the DHA-deficient animals more promptly utilized lipids for energy production than the wild-type mice (Fig. 2F). Taking these findings together, we can conclude that a higher lipid oxidation contributes to lower levels of fat accumulation in the Elovl2-ablated animals. High-fat diet is considered to be one of the main stressors that can induce negative effects in animals such as metabolic syndrome with obesity, insulin resistance, and fatty liver. In conjunction with this, it has been reported that a high-fat diet regime increases the level of hepatic lipogenesis and induces hepatic steatosis via upregulation of SREBP-1c (22Lin J. Yang R. Tarr P.T. Wu P.H. Handschin C. Li S. Yang W. Pei L. Uldry M. Tontonoz P. et al.Hyperlipidemic effects of dietary saturated fats mediated through PGC-1beta coactivation of SREBP.Cell. 2005; 120: 261-273Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, 27Biddinger S.B. Almind K. Miyazaki M. Kokkotou E. Ntambi J.M. Kahn C.R. Effects of diet and genetic background on sterol regulatory element-binding protein-1c, stearoyl-CoA desaturase 1, and the development of the metabolic syndrome.Diabetes. 2005; 54: 1314-1323Crossref PubMed Scopus (198) Google Scholar). To further investigate how DHA-deficient Elovl2-ablated animals, which already have induced hepatic levels of SREBP-1c, respond to this kind of stress condition, animals were given a high-fat (40%) diet treatment for 12 weeks. Although the consumption of high-fat diet affected the lipid composition in both liver and serum (supplementary Tables III–V), the relative differences in the PUFA profile between Elovl2−/− and control mice, seen under standard chow diet conditions (Fig. 1A–C), remained (Fig. 3A–C). In accordance with the expected findings, wild-type mice on high-fat diet compared with standard chow diet (set to 1) showed elevated levels of both SREBP-1c mRNA (Fig. 3D) and nSREBP-1c protein (Fig. 3E) in liver. Although the treatment with high-fat diet induced SREBP-1c mRNA levels even further in Elovl2−/− mice, there was no significant difference in absolute values between Elovl2−/− and wild-type mice (Fig. 3D). However, Elovl2-ablated animals showed higher levels of nSREBP-1c protein after the treatment compared with wild-type littermates (Fig. 3E). Interestingly, and in contrast to the data from Elovl2−/− animals maintained on standard chow diet, upregulation of nSREBP-1c by high-fat diet did not result in increased, but rather in decreased, expression of the genes of lipogenic enzymes FAS and SCD1 (Fig. 3D) in both wild-type and Elovl2−/− mice, suggesting an SREBP-1c-independent mechanism in the control of lipogenesis under these conditions. Surprisingly, despite superinduced levels of nSREBP-1c by high-fat diet, Elovl2−/− animals did not show any signs of fatty liver (Fig. 3F), and the TG levels were significantly lower than in wild-type mice (Fig. 3G), which again accentuates the observation that reduced levels of DHA in these mice are protective against fatty liver. In accordance with reduced TG levels in liver, Elovl2−/− mice maintained on high-fat diet did not gain as much weight in comparison with their wild-type littermates (Fig. 3H). This difference was mainly due to lower fat mass in the Elovl2−/− mice measured at two different time points (Fig. 3I), where the first 4 weeks of high-fat diet treatment seemed to be the most dramatic period when the Elovl2−/− animals showed almost no increase in weight or fat gain in comparison with their wild-type littermates. No difference in food intake was observed between KO and control littermates (Elovl2−/− 389.5 ± 7.5 vs. wild type 384.6 ± 5.7 kJ/week). One possible explanation for the discrepancy in weight gain between short-term and long-term hig
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