Induction of peroxisomal lipid metabolism in mice fed a high-fat diet

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1 MOLECULAR MEDICINE REPORTS 4: , 2011 Induction of peroxisoml lipid metbolism in mice fed high-ft diet SACHI KOZAWA 1,4, AYAKO HONDA 1, NAOMI KAJIWARA 1, YASUHIKO TAKEMOTO 1, TOMOKO NAGASE 1, HIDEKI NIKAMI 2, YUKIO OKANO 3, SHIGERU NAKASHIMA 4 nd NOBUYUKI SHIMOZAWA 1 Divisions of 1 Genomic Reserch, nd 2 Animl Experiment, Life Science Reserch Center, Gifu University; 3 Gifu University (trustee); 4 Deprtment of Cell Signling, Division of Moleculr nd Cellulr Biology, Gifu University Grdute School of Medicine, Gifu , Jpn Received April 1, 2011; Accepted August 8, 2011 DOI: /mmr Abstrct. Peroxisomes ctlyze rnge of essentil metbolic functions, minly relted to lipid metbolism. However, their roles in obesity hve yet to be clrified. The im of this study ws to investigte the correltion between obesity nd peroxisoml lipid metbolism, prticulrly very long-chin ftty cid (VLCFA) metbolism, gene expression of peroxisoml β-oxidtion enzymes, peroxisoml ATP binding cssette (ABC) trnsporter drenoleukodystrophy (ABCD1) gene nd its relted gene, ABCD2, the elongtion of the VLCFA (ELOVL) gene fmily nd the trnscriptionl fctors involved in the regultion of these genes, including peroxisome prolifertor-ctivted receptor α (PPARα) nd sterol regultory element binding protein. These fctors were nlyzed in livers from mice fed high-ft diet (HFD) or regulr diet (RD) for 20 weeks. Furthermore, the mounts of plsm sturted nd unsturted ftty cids, including VLCFAs, were mesured. A HFD induced heptic gene expression of not only hydroxysteroid 17-β dehydrogense 4 (HSD17b4) nd sterol crrier protein 2 (SCP2) in peroxisoml β-oxidtion enzymes but lso of ELOVL1, 2, 5 nd 6, which re involved in the elongtion of sturted nd unsturted VLCFAs. Furthermore, ABCD2 mrna prominently incresed in the HFD mice. The trnscriptionl regultor of these genes, PPARα, ws lso up-regulted in the HFD mice. VLCFA rtios including C24:0/ C22:0, C25:0/C22:0 nd C26:0/C22:0 re the most significnt dignostic mrkers of inherited peroxisoml diseses. These rtios were found to be low in the plsm of the HFD mice compred with the RD mice. The results suggest tht HFD ctivtes heptic peroxisoml VLCFA metbolism, nd my provide useful fundmentl informtion to explin the role of peroxisoml function in obesity nd lifestyle-relted diseses. Correspondence to: Dr Nobuyuki Shimozw, Division of Genomic Reserch, Life Science Reserch Center, Gifu University, 1-1 Yngido, Gifu , Jpn E-mil: nshim@gifu-u.c.jp Key words: obesity, peroxisoml bet oxidtion, very long-chin ftty cids Introduction Peroxisomes re single-membrne-lined orgnelles present in ll eukryotic cells, which ctlyze rnge of essentil metbolic functions including β-oxidtion of VLCFA nd the synthesis of bile cids nd plsmlogen in mmmls. Inborn errors of peroxisoml metbolism in humns, cusing peroxisoml diseses, consist minly of two lrge groups. The first group comprises peroxisoml biogenesis disorders (PBDs), which re chrcterized by fetl utosoml recessive diseses with no effective therpy. These disorders re cused by defect in PEX genes, which encode peroxins, proteins necessry for the biogenesis of peroxisomes nd the import of the peroxisoml mtrix nd membrne proteins, resulting in the generlized loss of metbolic function of peroxisomes. The second group includes diseses with single enzyme deficiencies, including peroxisoml β-oxidtion such s stright-chin cyl CoA oxidse (ACOX1) nd D-bifunctionl protein (DBP). X-linked drenoleukodystrophy (ALD) is the most common peroxisoml disese, with impired β-oxidtion of sturted VLCFA resulting in the ccumultion of VLCFA in tissues nd plsm. ALD is cused by defect of the ABCD1 gene, which encodes the peroxisoml ABC trnsporter ALD protein. Since most of these peroxisoml diseses with β-oxidtion dysfunction revel the ccumultion of VLCFA, incresed VLCFA in the plsm hs been used s dignostic mrker for these diseses (1). The genes encoding the peroxisoml β-oxidtion pthwy in the liver re trnscriptionlly regulted by PPARα, which is nucler hormone receptor (2). PPARα plys key role in energy expenditure, nd its substrtes involved in ftty cid oxidtion contining VLCFA my function s PPARα lignds (3). On the other hnd, it hs recently been shown tht dministrtion of HFD in mice resulted in the up-regultion of heptic PPARα mrna (4). These results suggest the possible involvement of peroxisoml metbolism in obesity. Therefore, the regultion of PPARα ctivity through modulting endogenous lignds my hve the potentil to generte new therpeutic pproch for obesity nd lifestyle-relted diseses (5). In the present study, in order to obtin insight into the role of peroxisoml metbolism, prticulrly VLCFA metbolism in obesity, we compred the levels of VLCFAs

2 1158 KOZAWA et l: PEROXISOMES IN HIGH-FAT DIET MICE in plsm. We lso compred the mrna levels of the genes involved in peroxisoml β-oxidtion, ALD nd ALD-relted protein (ABCD1 nd 2), nd elongtion of VLCFAs long with their regultor genes, PPARα, retinoid X receptor α (RXRα) nd sterol regultory element-binding protein (SREBP), in the livers of HFD-feeding nd regulr diet (RD)-feeding mice. Mterils nd methods Mice nd diets. C57BL/6J Jcl femle mice in their 14th dy of pregnncy were obtined from Cle Jpn Inc. (Tokyo, Jpn) nd housed in two groups: one group ws fed on HFD (HFD32, Cle Jpn Inc.) nd the other ws fed on RD (CE2, Cle Jpn Inc.). The mice were llowed ccess to wter nd HFD or RD (for diet composition see Tble Ⅰ). The regimens were mintined following delivery of the pups. Following wening (t dy 21), femles were excluded from further study, nd we nlyzed only mle pups in the two groups fed on RD nd HFD. The body weight of mice in this study ws mesured every 2 or 3 weeks. The nimls were mintined under controlled light (12-h light/drk cycle, light on t 08:00) nd temperture (23±2 C) conditions. All of the niml cre nd experimentl procedures were conducted under the regultions for niml experiments of Gifu University. Blood collection for biochemicl nlysis. Blood smples (0.3 ml) were drwn t week 6 nd 20 from the submndibulr veins of the mice with goldenrod niml lncet (MEDI Point, NY, USA) in 1.5 ml tube with 5 µl cogulnt (Wko, Jpn). Ethics nd experimentl procedures were pproved by the Committee for Animl Reserch nd Welfre of Gifu University. Smples were centrifuged t 2,500 x g for 15 min t room temperture nd plsm ws collected nd stored t -20 C until nlysis. Leptin nd diponectin levels in plsm from mice were mesured by Orientl Yest Co., Ltd. t week 20 using commercil kits for ELISA (Moring Institute of Biologicl Science Inc. nd Otsuk Phrmceuticl Co., Ltd, respectively). Mesurements of sturted nd unsturted ftty cids were tken, including rtios of C24:0/C22:0, C25:0/C22:0, C26:0/C22:0 nd C16:0 DMA (plsmlogen)/c16:0 in 50 µl plsm from mice, using gs chromtogrphy/mss spectrometry (GC/MS) s previously described (6). Tissue collection nd rel-time quntittive PCR. Mice were scrificed by cervicl disloction t week 20, nd the livers were removed nd preserved in RNAlter (Applied Biosystems, Tokyo, Jpn), nd stored t -20 C for RNA preprtion. Totl RNA ws purified with Isogen (Nippon Gene, Jpn) nd cdna ws synthesized with M-MLV reverse trnscriptse (Invitrogen, Jpn). Rel-time PCR nlysis ws crried out in n ABI PRISM 7000 using fluorescent TqMn methodology. Rel-time quntittive PCR ws performed for ech of the following genes, using redy-to use primer nd probe sets pre-developed by Applied Biosystems (TqMn gene expression ssys): ACOX1 (Mm _m1), ACOX2 (Mm _m1), HSD17b4 (Mm _m1), cetyl-coenzyme A cyltrnsferse 1A (ACAA1, Mm _s1), SCP2 (Mm _m1), ABCD1 (Mm _m1), ABCD2 Tble I. Ftty cids composition of experimentl diets. Regulr diet High-ft diet (CE2) (HFD32) Crude lipid content 4.63/100 g 31.9/100 g Composition of ftty cids 100% 100% C14: C14:1 0.3 C15:0 0.1 C16: C16: C17: C17:1 0.3 C18: C18: C18: C18: C20: C20: C20:4 C20: C22: C22: C24: C24: Unidentified Included in the unidentified composition. (Mm _m1), PPARα (Mm _m1), SREBP-1c (Mm _m1), Elovl1 (Mm _m1), Elovl2 (Mm _m1), Elovl3 (Mm _m1), Elovl4 (Mm _m1), Elovl5 (Mm _m1), Elovl6 (Mm _s1), Elovl7 (Mm _m1), leptin receptor (LEPR, Mm _m1), tumor necrosis fctor (TNF, Mm _m1), diponectin (Adipoq, Mm _m1), diponectin receptor 1 (Adipor1, Mm _mH), Adipor2 (Mm _g1), monocyte chemottrctnt protein 1 (MCP1, Mm _m1), uncoupling protein 2 (UCP2, Mm _m1), ftty cid synthse (FASN, Mm _m1), RXRα (Mm _m1), thyroid hormone receptor β (THRβ, Mm _m1) nd eukryotic 18S rrna (18S, Hs _s1) s n endogenous control. The mrna Ct vlues for these genes were normlized to eukryotic 18S rrna nd expressed s reltive increse or decrese in the liver from the HFD mice to those in the RD mice. Sttisticl nlysis. Results re expressed s the mens ± stndrd devition (SD). Sttisticl nlysis ws performed using the F-test nd two-tiled t-test for RT-PCR nd the F-test nd one-tiled t-test for other experiments.

3 MOLECULAR MEDICINE REPORTS 4: , A B C Figure 1. Body weight chnge, plsm leptin nd diponectin levels in regulr nd HFD mice. (A) The mens ± SD of body weight chnge. (B) Plsm leptin level (Lep) (ng/ml). (C) Plsm diponectin level (ADN) (mg/ml). Sttisticlly significnt differences by the Student's t-test re shown by *** p<0.005 RD vs. HFD. A B C D E F G H Figure 2. Plsm VLCFA levels in the RD nd HFD mice. The rtios of (A nd E) C24:0/C22:0, (B nd F) C25:0/C22:0, (C nd G) C26:0/C22:0 nd (D nd H) C16:0 DMA/C16:0 in plsm from RD nd HFD mice t weeks 6 nd 20, respectively. Sttisticlly significnt differences using the Student's t-test re shown by * p<0.05, ** p<0.01, *** p<0.005, RD vs. HFD, respectively (w, weeks). Results Body weight gin, plsm leptin nd diponectin levels. Growth curves of mice fed with HFD (n=8) or RD (n=7) re shown in Fig. 1A. At week 20, body weight of the HFD mice ws prominently incresed compred to tht of the RD mice (RD, 28.08±1,153 g; HFD, 45.03±5,081 g, p<0.005). The plsm leptin level of the HFD mice ws lso mrkedly incresed compred to tht of the RD mice (Fig. 1B), wheres no difference ws observed in plsm diponectin levels between HFD nd RD (Fig. 1C). VLCFA. We compred the rtios of C24:0/C22:0, C25:0/C22:0 nd C26:0/C22:0 in plsm between the HFD nd RD mice t weeks 6 nd 20 (HFD, n=8; RD, n=5 nd n=7, t weeks 6 nd 20, respectively). C24:0/C22:0 nd C25:0/C22:0 rtios in the HFD mice t week 6 were significntly lower thn those in RD mice (Fig. 2A nd B), nd three VLCFA rtios in the HFD mice t week 20 were significntly lower compred to those in the RD mice (Fig. 2E-G). By contrst, plsmlogen content (C16:0 DMA/C16:0) in the HFD mice ws higher thn tht of the RD mice t both 6 nd 20 weeks (Fig. 2D nd H). These results suggest tht peroxisoml lipid metbolism, including

4 1160 KOZAWA et l: PEROXISOMES IN HIGH-FAT DIET MICE Tble Ⅱ. Sturted nd unsturted ftty cids in plsm from feeding HFD nd RD mice t 20 weeks of ge. RD (verge) n=7 HFD (verge) n= µg/50 µl plsm µg/50 µl plsm C14: C14: C16:0 DMA C16: C16: C18: C18: C18: b b C18: C20: C20: ARA EPA C22: b b C22: C24: C24: DHA C25: C26: b b Sttisticlly significnt differences by the Student's t-test re shown by p<0.005, b p<0.05, respectively. C16:0 DMA, plsmlogen; ARA, rchidonic cid; EPA, eicospentnoic cid; DHA, docoshexenoic cid. β-oxidtion of VLCFA nd plsmlogen biosynthesis, were ctivted in the HFD mice. Further results including levels of sturted nd unsturted ftty cids in the HFD nd RD mice t week 20 re shown in Tble Ⅱ. Quntittive rel time PCR. Concerning peroxisoml β-oxidtion enzymes, the heptic gene expression of HSD17β4 nd SCP2 ws significntly up-regulted in the HFD mice t week 20, s compred to the RD mice. Furthermore, peroxisoml ABCD2 mrna ws mrkedly higher in the HFD mice (Fig. 3A). Among the ELOVL gene fmily, ELOVL1, 2, 5 nd 6 were up-regulted in the HFD mice (Fig. 3B). The level of ELOVL7 mrna ws elevted 12-fold in the HFD mice, lthough the chnge ws not sttisticlly significnt. Additionlly, mong the regultory genes of these enzyme genes, PPARα incresed significntly in the HFD mice. The SREBF regulting gene involved in ftty cid synthesis reveled tendency to increse in the HFD mice, but this ws not sttisticlly significnt. Further rel time PCR nlysis of heptic gene expression reveled tht mrna levels of TNF, Adipor2, MCP1, UCP2 nd FASN were elevted, nd tht of LEPR ws significntly down-regulted in the HFD mice, s compred to the RD mice (Fig. 3C). Discussion Peroxisomes re involved in number of essentil functions in mmmls, minly relted to lipid metbolism, including ftty cid β-oxidtion, plsmlogen biosynthesis nd phytnic cid α-oxidtion. The significnce of peroxisomes for humns is stressed by the existence of n expnding group of humn diseses in which peroxisome function is impired (7). The peroxisoml β-oxidtion metbolizes substrtes including VLCFAs such s C24:0 nd C26:0, pristnic cid nd the bile cid intermedites. Inborn errors of peroxisoml metbolism re minly divided into PBDs nd single peroxisoml enzyme deficiencies (1). Most of these peroxisoml diseses involve β-oxidtion dysfunction. Therefore, the ccumultion of VLCFAs in the plsm hs been used s dignostic mrker for these diseses (1). Furthermore, we hve estblished dignostic system for peroxisoml diseses in Jpn, using mesurements of incresed VLCFA nd phytnic cid, nd decresed plsmlogen in plsm from the ptients (6). A nucler trnscription fctor, PPARα, forms heterodimer with the RXR nd binds to trget genes involved in peroxisome prolifertion nd ftty cid metbolism, widely including β-oxidtion. HFD is the mjor cuse of obesity nd in wildtype HFD mice it ws ssocited with significnt increse in heptic PPARα gene expression. Activtion of PPARα leds to n increse in tissue-specific expression of the key genes involved in ftty cid uptke nd β-oxidtion (4,8). To elucidte the role of peroxisoml lipid metbolism in obesity, we compred HFD nd RD mice. The dms in the HFD group were dministered HFD t dy 14 of their pregnncy. The HFD mice gined body weight incresingly s compred with the RD mice (Fig. 1A). The plsm leptin level in the HFD mice ws significntly higher (pproximtely 14-fold) thn tht in RD mice, wheres no difference in diponectin levels ws observed between the HFD nd RD mice (Fig. 1B nd C). Of note, HFD induced decrese in VLCFA levels (C24:0/ C22:0, C25:0/C22:0 nd C26:0/C22:0) nd n increse in plsmlogen levels (C16:0DMA/C16:0) in plsm, compred with the RD (Fig. 2), suggesting the possible ctivtion of heptic peroxisoml function by HFD. Therefore, mrna expression levels of peroxisoml β-oxidtion enzymes including ACOX1, HSD17β4, AACA1, nd their regultory genes, PPARα, SREBP, MCP1 nd UCP2 were nlyzed. Most of the heptic mrnas involved in peroxisoml β-oxidtion, nd lso their regultory genes, were up-regulted in the HFD mice compred with the RD mice (Fig. 3). These results indicte tht HFD my induce peroxisoml metbolism in the liver. Furthermore, the mrna expression level of ABCD2 incresed 6-fold in the HFD mice. ABCD2 is speculted to ct s dimeriztion prtner of ABCD1, the X-linked drenoleukodystrophy-ssocited protein (9), nd hs functionl redundncy with ABCD1. Therefore, mrna expression levels of RXRα nd THRβ, s well s PPARα nd SREBP, which were reported to be regultors of ABCD2 expression were ssessed (10). Only PPARα mrna expression ws found to be higher in the HFD mice thn in the RD mice (Fig. 3). The ELOVL gene fmily comprises of 7 distinct ftty cid elongse subtypes (Elovl1-7). Ech elongse hs distinct tissue distribution, nd the individul enzymes exhibit

5 MOLECULAR MEDICINE REPORTS 4: , A B C Figure 3. Heptic gene expression in RD nd HFD mice. Dt were clculted reltive to the expression of 18S rrna. The F-test nd two-tiled t-test re indicted by * p<0.05, ** p<0.01, *** p<0.005, RD vs. HFD, respectively (w, weeks). different ftty cid substrte preferences. The metbolic pthwys of long-chin ftty cids ply significnt role in the membrne lipid composition s well s in the genertion of precursors for cell signling molecules. In this study, Elovl7 mrna ws up-regulted 12-fold, nd levels of Elovl1, 2, 5 nd 6 were lso higher in the HFD mice (Fig. 3B). Elovl7 expression is regulted by lipogenic enzymes nd the ndrogen pthwy through SREBP (11). Only Elovl3 reveled decresing tendency in the HFD mice (Fig. 3B). Brolinson et l (12) reported similr results, reveling tht Elovl3 expression ws found to be suppressed in ABCD2 overexpressing mice. A HFD is the mjor cuse of obesity, which increses oxidtive stress ccompnied by n elevtion of NADPH oxidse expression. Production of selective rective oxygen species in the dipose tissue of obese mice leds to the development of inflmmtory processes nd to the dysregultion of dipocytokines, including diponectin, plsminogen ctivtor inhibitor-1 nd IL-6 (13). Furthermore, vriety of dietry components my ply significnt roles in the development of insulin resistnce (14), which my ffect the progression of dibetes (15), heptic stetosis (16), crdiovsculr disese nd hypertension (17). Peroxisoml lipid metbolism hs emerged s n essentil pthwy relted to lifestyle-relted diseses, including obesity, dibetes nd crdiovsculr disese. Gining further insight into the peroxisoml metbolic pthwy my provide useful informtion to develop new therpeutic pproches to these diseses. The dt reveling peroxisoml metbolites nd gene expression in the HFD mice my provide useful informtion for the understnding of these lifestyle-relted diseses. Acknowledgements The uthors thnk Dr Y. Okzki nd Dr T. Imnk for their helpful comments. This study ws supported by grnt-in-id for Scientific Reserch ( ) from the Jpn Society for the Promotion of Science, grnt for Child Helth nd Development nd grnt for Reserch on Intrctble Diseses from the Helth nd Lbor Sciences Reserch Grnts.

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