Role of Nrf2 in the alteration of cholesterol and bile acid metabolism-related gene expression by dietary cholesterol in high fat-fed mice

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1 Originl Article JCBN Journl the Kyoto, jcbn /jcbn Originl Society Jpn of Article Clinicl for Free Biochemistry Rdicl Reserch nd Nutrition Jpn Role of Nrf2 in the ltertion of cholesterol nd bile cid metbolism-relted gene expression by dietry cholesterol in high ft-fed mice Toshinori Kmisko, 1, * Yuji Tnk, 1 Yoshizumi Kishino, 1,2 Tknori Iked, 3 Kzuo Ymmoto, 4 Shiori Msud 1 nd Hiroshi Ogw 3 1 Deprtment of Clinicl Lbortory Medicine, Kinki University Fculty of Medicine, Osksym, Osk , Jpn 2 Deprtment of Biomedicl Informtics, Division of Helth Sciences, Osk University Grdute School of Medicine, Suit, Osk , Jpn 3 Fculty of Humn Sciences, Tezukym Gkuin University, Ski, Osk , Jpn 4 Division of Bsic Medicl Science, Kinki University Fculty of Medicine, Osksym, Osk , Jpn (Received?? 21 October, 2013; Accepted 4 December, 2013; Published online 14 Februry, 2014) Nucler Cretive stricted vided Copyright 2014 This the is use, fctor-e2-relted originl Commons n open distribution, 2014 work ccess JCBN Attribution rticle nd properly fctor reproduction distributed 2 License, cited. (Nrf2) is under which in ny regultor the permits medium, terms of of unre- lipid pro- the metbolism s well s vrious cytoprotective enzymes nd my be involved in the pthogenesis of non-lcoholic ftty liver disese. Although, bile cids ffect lipid metbolism, the role of Nrf2 in bile cid metbolism remins uncler. In this study, it ws tested how Nrf2 modultes lipid nd bile cid homeostsis in liver in response to chnges of cholesterol bsorption under high-ft diet using Nrf2-null mice. Eight-week-old mle wild-type nd Nrf2-null mice (n = 6/group) were divided into three groups fed the following diets: 1) control diet contining 4% soyben oil nd 16% lrd, 2) control diet plus ezetimibe, 3) control diet plus cholesterol. Blood nd livers were removed fter 4 weeks feeding. High cholesterol diet incresed heptic expression of liver X receptor α trget genes relted to ftty cid metbolism (FAS, ACC1, SREBP-1c, SCD-1c nd CD36), cholesterol trnsport (Abcg5/ bcg8) nd bile cid synthesis (Cyp71) in wild type mice. However, these genes were not induced in Nrf2-null mice. These findings suggest tht Nrf2 hs reltion to liver X receptor α nd controls the regultion of gene expressions relted to lipid nd bile cid metbolism. Key Words: nucler fctor-e2-relted fctor 2, non-lcoholic ftty liver disese, cholesterol metbolism, bile cid metbolism, liver X receptor α H Introduction High ft diet is well-known contributor to the pthogenesis of non-lcoholic ftty liver disese (NAFLD). NAFLD represents the heptic mnifesttion of the metbolic syndrome, chrcterized by insulin resistnce, dyslipidemi, nd hypertension. (1,2) The term NAFLD contins brod spectrum of liver diseses rnging from simple stetosis to non-lcoholic stetoheptitis (NASH). (3,4) Oxidtive stress hs been suggested to be importnt for the progression from simple stetosis to NASH nd severl studies hve investigted the efficcy of ntioxidtive supplements ginst progression of NASH. (5,6) Recent studies showed tht dietry cholesterol is importnt for the progression of heptic stetosis. (7 10) Ezetimibe is n inhibitor of the cholesterol uptke trnsporter, Niemnn-Pick C1-like 1 (NPC1L1), loctes in the picl membrne of enterocytes. (11,12) Severl studies showed tht ezetimibe ttenutes liver stetosis in experimentl models. (13,14) These findings suggest tht the mngement of cholesterol intke is one of the therpeutic trgets for NAFLD. In ddition to the cholesterol intke nd biosynthesis, ctbolism to bile cids is lso criticl for mintining cholesterol homeostsis. Administrtion of nturl (colic cid) or synthetic (GW4064) lignd for the nucler bile cid receptor frnesoid X receptor (FXR) is known to prevent high-ft diet-induced heptic stetosis in mice. (15,16) The Nucler fctor-e2-relted fctor 2 (Nrf2), which is cellulr sensor for oxidtive nd electrophilic stress, induces the vrious cytoprotective enzymes. (17) Recent studies showed tht Nrf2 deletion ffected lipid metbolism. Nrf2-null mice showed the dysregultion of β-oxidtion nd the increse in heptic triglycerides. (18,19) In Nrf2-null mice, the methionine- nd cholinedeficient diet cused rpid onset nd progression of nutritionl stetoheptits. (20) Furthermore, Nrf2 ctivtor prevented high-ft diet induced heptic lipid ccumultion in wild type mice but not in Nrf2-null mice. (21) Nrf2 my ply role in lipid metbolism nd my be involved in the pthogenesis of NAFLD. Nrf2 lso regultes heptic bile cid metbolism. Sulforphne, n Nrf2 ctivtor induced the heptic FXR mrna expression in wild-type mice but not in Nrf2-null mice. (22) The purpose of this study is to evlute the role of Nrf2 on bile cid s well s lipid metbolism in high ft diet induced NAFLD model. In this study, we investigted the effect of cholesterol bsorption on lipid nd bile cid metbolism in Nrf2-null mice nd wild type mice. To chnge the cholesterol bsorption, mice were fed diet contining high cholesterol or ezetimibe. In humn nd rt, NPC1L1 is expressed both in intestine nd liver. Therefore, ezetimibe my ffect heptic lipid metbolism. On the other hnd, NPC1L1 is predominntly expressed in the intestine in mice nd is hrdly expressed in the liver. (11) Ezetimibe only inhibits intestinl cholesterol bsorption nd does not directly ffect heptic lipid metbolism in mice. Here, we reported tht the induction of the gene expression relted to lipid nd bile cid metbolism by cholesterol influx ws t lest prtilly controlled by Nrf2. Mterils nd Methods Mterils nd chemicls. Milk csein, corn strch, α-corn strch, minerl AIN-93 mixture nd vitmin AIN-93 VX mixture were purchsed from CLEA Jpn (Osk, Jpn). Soyben oil nd lrd were purchsed from Orientl Yest (Tokyo, Jpn) nd Ymkei (Osk, Jpn), respectively. Ezetimibe ws kindly provided by Schering-Plough Co., Ltd. (Osk, Jpn). All other chemicls were purchsed from Wko Pure Chem. Ind., Ltd. (Osk, Jpn), unless noted. *To whom correspondence should be ddressed. E-mil: kmisko@med.kindi.c.jp doi: /jcbn J. Clin. Biochem. Nutr. Mrch 2014 vol. 54 no

2 Tble 1. Serum nd heptic concentrtions of lipids nd bile cids in wild-type nd Nrf2-null mice Wild-type mice Nrf2-null mice Control EZ CH Control EZ CH Serum concentrtion Triglycerides (mg/dl) 27 ± 2 19 ± 3 15 ± 2 19 ± 3 23 ± 2 13 ± 1 Cholesterol (mg/dl) 144 ± ± ± ± 14 c 101 ± ± 16 HDL-cholesterol (mg/dl) 79 ± 5 58 ± 3 72 ± 8 55 ± ± ± 8 Bile cids (μmol/l) 4.6 ± ± ± ± ± ± 1.4 Heptic concentrtion Triglycerides (mg/g liver) 22 ± 4 9 ± 1 66 ± 6 45 ± ± 2 68 ± 10 Cholesterol (mg/g liver) 2.5 ± ± ± 2.0 b 3.3 ± ± ± 0.6 b Bile cids (μmol/g liver) ± ± ± ± ± ± p<0.05 significnt difference from mice fed with control diet. b p<0.01 significnt difference from mice fed with control diet. c p<0.05 significnt difference between genotypes. Animls nd diets. A colony of wild-type nd Nrf2-null mice, generted originlly by Itoh et l. (23), were bckcrossed with C57BL/6 mice for ten genertions. All mice were housed in the sme niml cre fcility controlling for temperture (22 ± 2 C), humidity (55 ± 5%), nd light (lights on; 07:00 19:00). Eightweek-old mle wild-type nd Nrf2-null mice (n = 6/group) were divided into three groups fed the following diets: 1) the control diet group (C); contining 4% soyben oil,16% lrd, 14% milk csein, 0.18% L-cystine, 3.5% AIN-93G minerl mixture, 1% AIN-93VX vitmine mixture, 5% cellulose powder, 30.57% corn strch, 15.5% α-corn strch, 10% sucrose, 0.25% choline bitrtrte for 4 weeks; 2) the ezetimibe group (EZ); control diet contining ezetimibe (0.3 mg/dy) for 4 weeks; 3) the cholesterol diet group (CH); contining 4% soyben oil, 16% lrd nd 1% cholesterol, 14% milk csein, 0.18% L-cystine, 3.5% AIN-93G minerl mixture, 1% AIN-93VX vitmine mixture, 5% cellulose powder, 29.57% corn strch, 15.5% α-corn strch, 10% sucrose, 0.25% choline bitrtrte, 1% cholesterol, for 4 weeks. After 4 weeks, food ws removed 2 h prior to collecting tissues nd blood smples were collected by hert puncture fter nesthesi with pentobrbitl (50 mg/kg, intr-peritonelly), nd livers nd duodenum were hrvested nd stored t 80 C until use. Studies were pproved by Kinki University Fculty of Medicine Animl Cre nd Use Committee. Biochemicl nd histologicl nlysis. Serum biochemicl mrkers were quntified using biochemistry utonlyzer Lbospect 008 (Hitch High-Technologies Corportion, Ibrgi, Jpn). Liver lipid content ws extrcted ccording to the method of Folch et l. (24) Heptic cholesterol nd triglyceride concentrtions were determined by stndrd enzymtic-colorimetric ssys using commercilly vilble kits (Wko Pure Chem. Ind.). Heptic bile cid concentrtions were quntified. (25) In brief, 100 mg of liver ws homogenized in 900 μl of 1:1 t-butyllcohol/double distilled wter (v/v) nd incubted overnight t room temperture. The homogente ws centrifuged t 5,000 rpm for 10 min. Heptic bile cid levels were mesured in the superntnt by enzymticcolorimetric ssys using bile cid ssy kits (Wko Pure Chem. Ind.). Liver tissues were fixed in 10% prformldehyde, embedded in prffin nd stined with hemtoxylin-eosin. RNA isoltion nd rel-time polymerse chin rection. Totl RNA ws isolted using TRIzol regent (Life Technologies, Tokyo, Jpn) ccording to the mnufcturer s protocol. The concentrtion of totl RNA in ech smple ws quntified spectrophotometriclly t 260 nm. The mrna expression of lipid nd bile cid metbolism-relted genes nd 18s rrna were quntified by SYBR rel-time polymerse chin rection (PCR) (SYBR Premix Ex Tq; Tkr bio Inc., Shig, Jpn). Primers were used from previous reports. (18,26 34) The mplifiction rections were crried out in n ABI Prism 7900 HT sequence detection system (Applied Biosystems, Foster, CA). The mount of mrna ws clculted using the comprtive CT method which determines the mount of trget normlized to n endogenous reference. Ech gene ws normlized to 18s rrna. Sttisticl nlysis. Sttisticl nlysis ws performed using the softwre pckge, SYSTAT, ver. 11 (Systt Inc., Evnston, IL). Comprisons mong the groups were nlyzed by nlysis of vrince, followed by Tukey s multiple rnge test. All results re expressed s mens ± SEM. Significnce ws set t p<0.05. Results Serum nd heptic concentrtions of lipids nd bile cids in wild-type nd Nrf2-null mice. Lipid nd bile cid profiles in wild-type nd Nrf2-null mice re shown in Tble 1. Serum cholesterol ws higher in wild-type mice compred to Nrf2-null mice feeding the control diet. Ezetimibe decresed serum cholesterol nd HDL-cholesterol in wild-type mice. Serum triglyceride nd bile cids were not chnged in ech group fter feeding the respective diet. Heptic triglycerides tended to be higher in Nrf2- null control mice compred to wild-type control mice. Ezetimibe decresed heptic triglyceride both in wild-type mice nd Nrf2- null mice. Heptic cholesterol concentrtion ws not chnged by ezetimibe dministrtion nd ws significntly incresed by high cholesterol diet both in wild-type mice nd Nrf2-null mice. There were no significnt differences in heptic bile cid concentrtions between genotypes of ech group. Histologicl findings of liver in wild-type nd Nrf2-null mice. Histologicl findings of liver in wild-type nd Nrf2-null mice re shown in Fig. 1. Consistent with heptic triglyceride level, lipid droplets in heptocytes tended to increse in Nrf2-null control mice compred to wild-type control mice. Ezetimibe decresed heptic stetosis both in wild-type mice nd Nrf2-null mice. Heptic stetosis level ws incresed by cholesterol feeding both in wild-type mice nd Nrf2-null mice. Heptic expression of Nrf2 trget genes nd lipid metbolism-relted genes in wild-type nd Nrf2-null mice. The chnges in Nrf2 trget genes nd lipid metbolism-relted genes expression re summrized in Tble 2. In regrd to Nrf2 trget genes, heptic expression of glutmte-cysteine ligse, ctlytic subunit (Gclc) nd heme oxygense-1 (Ho-1) were mesured. Heptic gene expression of Gclc ws lower in Nrf2-null mice with cholesterol diet compred to the CH group in wild-type mice. Heptic expression of Ho-1 ws unchnged mong the three groups of both mice. With respect to the heptic ftty cid synthesis, the expression of ftty cid synthse (Fs) nd cetyl coenzyme-a crboxylse-1 (Acc1) were unchnged mong the three groups of both mice. Heptic gene expression of sterol regultory element-binding protein (Srebp)-1c, steroyl-coa desturse(scd1)nd CD36, n ftty cid trnsporter, were higher in the CH group compred to the EZ group in wild-type mice, nd T. Kmisko et l. J. Clin. Biochem. Nutr. Mrch 2014 vol. 54 no. 2 91

3 Fig. 1. Hemtoxylin nd eosin stined liver sections from wild type nd Nrf2 null mice fed with three experimentl diets. Tble 2. Heptic expression of Nrf2 trget genes nd lipid metbolism relted genes in wild type nd Nrf2 null mice Wild type mice Gclc Ho 1 Pprα Acox Cpt1 Srebp 1c Fs Acc1 CD36 SCD1 Srebp 2 Hmg CoA R LXRα Abcg5 Abcg8 Control EZ 1.00 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.12 Nrf2 null mice CH 1.01 ± ± ± 0.19c 1.78 ± 0.32c 1.37 ± 0.40b 2.03 ± 0.40c 0.72 ± ± ± 0.33c 1.84 ± 0.29b 0.69 ± ± ± 0.15b 4.39 ± 1.11,c 4.52 ± 0.95,c Control EZ CH 0.50 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.05d 0.51 ± ± ± ± ± ± ± ± ± ± 0.04b 0.58 ± ± ± ± 0.26 p<0.01 significnt difference from mice fed with control diet. bp<0.05 significnt difference from mice fed with ezetimibe diet. p<0.01 significnt difference from mice fed with ezetimibe diet. dp<0.05 significnt difference between genotypes. c the expression of these mrna were unchnged mong the three groups of Nrf2-null mice. In regrd to the ftty cid oxidtion, heptic gene expression of peroxisome prolifertor-ctivted receptors (Ppr) α, crnitine plmitoyltrnsferse (Cpt) 1, nd cyl-coenzyme A oxidse (Acox) 1 were higher in the CH group thn the EZ group in wild-type mice, nd the mrna expressions of these genes were unchnged mong the three groups of Nrf2null mice. The expression of Srebp-2 ws lower in the CH group thn the EZ group in Nrf2-null mice. However, there were no differences in the expression of this gene mong the three groups in wild-type mice. The mrna expression of 3-hydroxy-3-methylglutryl coenzyme A-reductse (Hmg-CoA-R) ws unchnged mong the three groups of both genotypes. The mrna expression of liver X receptor α (LXRα) ws induced by feeding the cholesterol diet in wild-type mice, however, 92 it ws unchnged mong the three groups of Nrf2-null mice. The mrna expression of ATP-binding cssette sub-fmily G member 5 (Abcg5) nd ATP-binding cssette sub-fmily G member 8 (Abcg8), the trget genes of Lxrα, ws induced by cholesterol feeding in wild-type mice. On the other hnd, the expression of Abcg5 nd Abcg8 were only slightly induced by cholesterol feeding in Nrf2-null mice. Heptic expression of bile cid metbolism relted genes in wild type nd Nrf2 null mice. The chnges in bile cids metbolism-relted gene expression re summrized in Tble 3. In wild-type mice, heptic gene expression of Fxr nd smll heterodimer prtner (Shp) were higher in the CH group compred to the EZ group. In Nrf2-null mice, there were no differences in the expression of Fxr nd Shp mong the three groups. In regrd to the bile cid synthesis, heptic gene expression of cholesterol 7 doi: /jcbn.13 92

4 Tble 3. Heptic expression of bile cid metbolism-relted genes in wild-type nd Nrf2-null mice Wild-type mice Nrf2-null mice Control EZ CH Control EZ CH Fxr 1.00 ± ± ± 0.24 c 0.81 ± ± ± 0.04 Shp 1.00 ± ± ± 0.40 c 0.81 ± ± ± 0.24 Cyp ± ± ± 1.06 b,c 3.21 ± ± ± 0.29 Cyp8b ± ± ± 0.32 c 1.08 ± ± ± 0.13 Cyp ± ± ± ± ± ± 0.09 Cyp7b ± ± ± 0.27,d 0.86 ± ± ± 0.08 Ntcp 1.00 ± ± ± ± ± ± 0.14 Otp ± ± ± ± ± ± 0.18 Otp ± ± ± ± ± ± 0.14 Bsep 1.00 ± ± ± ± ± ± 0.07 Mrp ± ± ± 0.24,d 1.00 ± ± ± 0.07 Mrp ± ± ± 0.43 d 0.21 ± 0.03 e 0.16 ± 0.03 e 0.23 ± 0.04 e Bt 1.00 ± ± ± ± ± ± 0.11 p<0.05 significnt difference from mice fed with control diet. b p<0.01 significnt difference from mice fed with control diet. c p<0.05 significnt difference from mice fed with ezetimibe diet. d p<0.01 significnt difference from mice fed with ezetimibe diet. e p<0.05 significnt difference between genotypes. α-hydroxylse (Cyp71), sterol 12-α-hydroxylse (Cyp8b1) nd 25-hydroxycholesterol 7-α-hydroxylse (Cyp7b1) of wild-type mice were higher in the CH group compred to the EZ group nd in Nrf2-null mice there were no differences in the mrna expression of these genes mong the three groups. Heptic mrna expression of sterol 27-hydroxylse (Cyp271) ws unchnged in both genotypes of ech group. With respect to heptic bile cid trnsporters, the expression of N + -turocholte cotrnsporting polypeptide (Ntcp), orgnic nion-trnsporting polypeptide (Otp)11, Otp14 nd bile slt export pump (Bsep) were unchnged mong the three groups of both genotypes. Heptic multidrug resistnce-ssocited protein (Mrp) 2 nd Mrp3 gene expression of wild-type mice were higher in the CH group compred to the EZ group, though, in Nrf2-null mice there were no differences in the expressions of these mrna mong the three groups. Discussion In this study, we investigted the roles of Nrf2 on cholesterol nd bile cid metbolism under high ft diet by using Nrf2-null mice. Our results showed tht Nrf2 is necessry for the gene regultion relted to cholesterol nd bile cid metbolism. SREBP2 nd LXRα re the regultors for heptic cholesterol metbolism. (35,36) High cholesterol diet is known to induce the expression of Abcg5 nd Abcg8, which re trget of LXRα. (37) In this experiment, the expression level of Abcg5 nd Abcg8 were not incresed by cholesterol feeding in Nrf2-null mice. This finding suggests tht Nrf2 my be required for LXRα ctivtion. Administrtion of LXRα gonists lso induced heptic mrna expression of ftty cid metbolism relted genes, such s FAS, ACC1, SREBP-1c, SCD-1c nd CD36. (38 40) In ccordnce with this phenomenon, high cholesterol diet induced these genes in wild type mice. Though, in Nrf2-null mice these genes were not induced by high cholesterol diet. These findings indicte tht Nrf2 hs roles in regultion of the expression of LXRα relted genes nd controls ftty cid metbolism s well s cholesterol metbolism. Although the defects in the ltertion of LXRα relted gene expression were observed in Nrf2-null mice, high cholesterol feeding incresed heptic triglyceride level s well s heptic cholesterol levels in both genotypes. Some fctor other thn LXRα my control heptic lipid concentrtions. With respect to cholesterol synthesis, the regultion of HMG-CoA-R is medited by SREBP clevge-ctivting protein (SCAP)-SREBP2 pthwy. (35) As expected, in this experiment, mrna expression of SREBP2 nd HMG-CoA-R were decresed by high cholesterol diet in both genotypes. This finding suggests tht Nrf2 my not be involved in cholesterol synthesis regulted by SCAP-SREBP2 pthwy. Regrding bile cid synthesis, FXR plys centrl role in the regultion of Cyp71, rte-limiting enzyme of bile cid synthesis. Severl recent studies hve shown tht Nrf2 controls the gene expression level relted to bile cid synthesis. (41,42) Ky HY et l. (19) demonstrted tht Nrf2 hs some roles in FXR ctivtion. In mice but not in humn, high cholesterol diet induces bile cid synthesis becuse Cyp71 is LXRα trget gene. (43) Similr to their study, we lso observed the induction of heptic Cyp71mRNA by high cholesterol feeding in wild-type mice. However, high cholesterol diet did not ffect the expression of Cyp71 in Nrf2-null mice. This phenomenon suggests tht Nrf2 is required for the regultion of clssic pthwy of bile cid synthesis vi LXRα in mice. With respect to bile cid trnsporters, it hs been reported tht Nrf2 regultes MRP2 nd BSEP in humn heptocytes, (44,45) which excrete bile cids into bile. Recent study demonstrted tht humn MRP2 is induced by LXRα in HepG2 cells. (46) However, it hs never reported tht rodent Mrp2 ws induced by LXRα. In this study, we demonstrted tht Mrp2 ws decresed in the EZ group nd ws incresed in the CH group in wild-type mice, suggesting tht rodent Mrp2 my be possibly induced by LXRα. In conclusion, it ws tested how Nrf2 modultes lipid nd bile cid homeostsis in liver in response to cholesterol supplementtion under the high ft diet. Nrf2 deletion dysregultes the expression of heptic LXRα trget genes which re relted to lipid nd bile cid metbolism. Acknowledgments The uthors would like to thnk Yoko Sito nd Mriko Shibym for technicl ssistnce. This work ws prtly supported by Grnt-in-Aid for Scientific Reserch (No ) from the Jpn Society for the Promotion of Science. Conflict of Interest No potentil conflicts of interest were disclosed. T. Kmisko et l. J. Clin. Biochem. Nutr. Mrch 2014 vol. 54 no. 2 93

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