ARTICLE. Keywords AMPK. Cholesterol. Insulin resistance. Intestine. Isoflavones. Liver. LXRα. LXRβ. Mice. Soy protein

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1 Dietologi () 55: DOI.7/s ARTICLE Soy protein isoflvones ifferentilly regulte liver X reeptor isoforms to moulte lipi metolism n holesterol trnsport in the liver n intestine in mie M. González-Grnillo & K. R. Steffensen & O. Grnos & N. Torres & M. Korh-Anré & V. Ortíz & C. Aguilr-Slins & T. Jkosson & A. Díz-Villseñor & A. Loz-Vles & R. Hernnez-Pno & J.-Å. Gustfsson & A. R. Tovr Reeive: 3 Jnury /Aepte: 3 April /Pulishe online: 9 June # Springer-Verlg Astrt Aims/hypothesis Liver X reeptor (LXR)α regultes the genes involve in holesterol, ftty i n gluose metolism. Soy protein (SP) onsumption reues the hepti umultion of holesterol n triylglyerol, n improves M. González-Grnillo n O. Grnos ontriute eqully to this work. Eletroni supplementry mteril The online version of this rtile (oi:.7/s ) ontins peer-reviewe ut uneite supplementry mteril, whih is ville to uthorise users. M. González-Grnillo : O. Grnos : N. Torres : V. Ortíz : A. Díz-Villseñor : A. Loz-Vles : A. R. Tovr (*) Deprtmento Fisiologí e l Nutriión, Instituto Nionl e Cienis Méis y Nutriión, Vso e Quirog No 5, Tllpn, Méxio D.F. 4, Méxio e-mil: tovr.r@gmil.om K. R. Steffensen : M. Korh-Anré : T. Jkosson : J.-Å. Gustfsson Deprtment of Biosienes n Nutrition, Krolinsk Institutet, Stokholm, Sween C. Aguilr-Slins Deprtment of Enorinology n Metolism, Instituto Nionl e Cienis Méis y Nutriión Slvor Zuirán, Mexio City, Mexio R. Hernnez-Pno Deprtment of Experimentl Pthology, Instituto Nionl e Cienis Méis y Nutriión Slvor Zuirán, Mexio City, Mexio J.-Å. Gustfsson Center for Nuler Reeptors n Cell Signling, University of Houston, Houston, TX, USA insulin sensitivity. However, it is not known whether these effets re meite vi LXRα. We therefore investigte whether the onsumption of SP regultes metoli hnges in holesterol metolism n insulin sensitivity vi LXRα. Methos Wil-type (WT) n Lxrα / (Lxrα, lso known s Nrh3) mie were fe n SP iet with or without holesterol for 8 ys. The expression of LXRα trget genes ws mesure in liver n intestine, s were hepti lipi ontent n fel ile i onentrtion. Orl gluose n insulin tolerne tests were lso performe. Heptoytes were use to stuy the effet of isoflvones on LXR tivity. Results The livers of WT n Lxrα / mie fe n SP highholesterol iet showe less stetosis thn those fe sein. The SP iet inrese the expression of the ATP-ining ssette (ABC) su-fmily genes A, Ag5 n Ag8 in the liver n intestine, s well s inresing totl fel ile i exretion n insulin sensitivity in WT mie ompre with mie fe sein iet. However, these effets of SP were not oserve in Lxrα / mie. The SP isoflvone, genistein, represse the tivtion of LXRα trget genes y T937, wheres it stimulte the tivtion of LXRβ trget genes. The AMP-tivte protein kinse inhiitor, ompoun C, h the opposite effets to those of genistein. Conlusions/interprettion Our results suggest tht SP isoflvones stimulte the phosphoryltion of LXRα or LXRβ, resulting in ifferent iologil effets for eh LXR isoform. Keywors AMPK. Cholesterol. Insulin resistne. Intestine. Isoflvones. Liver. LXRα. LXRβ. Mie. Soy protein

2 47 Dietologi () 55: Arevitions ABC ATP-ining ssette ACC Aetyl-CoA roxylse ALT Alnine trnsminse AMPK AMP-tivte protein kinse ITT Insulin tolerne test LXR Liver X reeptor LXRE LXR response element RCT Reverse holesterol trnsport SP Soy protein WT Wil-type Introution Oesity, mjor puli helth prolem roun the worl [, ], is ssoite with severl metoli normlities, whih inlue hypertension, hyperholesterolemi, hypertriylglyerolemi n insulin resistne [3, 4], n le to type ietes n riovsulr isese [5 8]. Serum holesterol levels re linilly mnge through the use of sttins [9]. However, numer of ietry moifitions hve een suggeste to reue serum holesterol onentrtions []. Severl met-nlyses hve shown tht the onsumption of soy protein (SP) reues totl n LDL-holesterol [, ]. Stuies in experimentl nimls hve lso emonstrte tht SP reues loo lipis, hepti holesterol n triylglyerol [3 5], n lso inreses insulin sensitivity [6]. The preise mehnism y whih SP reues serum n hepti holesterol hs not een estlishe, lthough it hs een suggeste tht these effets our through n inrese in ile i exretion [7]. The inter-orgn holesterol flux n the synthesis of ile is from holesterol re in prt regulte y the trnsription ftor, liver X reeptor (LXR)α [8, 9]. LXRs re lign-tivte trnsription ftors tht elong to the nuler reeptor superfmily []. The LXR sufmily onsists of two isoforms, LXRα n LXRβ, whih form oligte heteroimers with the retinoi X reeptor n regulte gene expression y ining to LXR response elements (LXREs) in the promoter regions of their trget genes [], some of whih re involve in reverse holesterol trnsport (RCT) []. LXRα is highly unnt in the liver, intestine, kiney, ipose tissue n mrophges, wheres LXRβ is uiquitously proue [3]. However, LXRα is the ominnt isoform in the liver, the tivtion of whih inreses iliry holesterol seretion n limits holesterol sorption [4]. Reent eviene suggests tht LXRα tivity n e regulte y phosphoryltion [5, 6]. There is eviene tht isoflvones re wek ligns for ertin other nuler reeptors [7 9], ut it is not known whether the onsumption of SP or its isoflvones (minly genistein n izein) moultes trnsriptionl ontrol of LXRs or regultes the phosphoryltion of these nuler reeptors. Our im, therefore, ws to use wil-type (WT) n Lxrα / (Lxrα lso known s Nrh3) mie n investigte whether metoli hnges tht inrese ile i exretion fter the onsumption of n SP iet re meite vi LXRα, thus leing to the upregultion of genes involve in ile i synthesis n RCT in the liver n intestine. Our stuy lso sought to etermine whether isoflvones re le to tivte LXRα, iretly or iniretly, n whether LXRα n meite the trnsriptionl effets of SP on the regultion of genes involve in ftty i synthesis, RCT n insulin sensitivity. Methos Animls, iet formultion n feeing Mle Lxrα / mie were otine from Gustfsson s lortory.thesemie were krosse for ten genertions in C57BL/6J mie [3]. C57BL/6J ontrol mie were purhse from Toni Europe (Lille Skensve, Denmrk). Mle mie t 3 to 4 months of ge h free ess to wter n one of the experimentl iets. These isolori iets, the omposition n soures of whih re shown in eletroni supplementry mteril (ESM) Tle, were ministere in ry form. The isolte SP use in these stuies h 88% purity. The Lxrα / or WT mie were ivie into four experimentl groups s follows (n eh): () % sein; () % sein plus % holesterol; (3) % SP; n (4) % SP plus % holesterol. The nimls were house in miroisoltors with h light/ rk yle n reeive the experimentl iets for 8 ys. At the en of the stuy, the nimls were fste for 8 h, kille y ron ioxie inhltion n epitte. The loo ws ollete n serum, otine y entrifugtion t, g, store t 7 C until further nlysis. Liver, ileum n gll ler were frozen in liqui nitrogen n store t 7 C until further nlysis. The niml protool ws pprove y the Animl Committee of the Ntionl Institute of Meil Sienes n Nutrition, Mexio City. Cholesterol n triylglyerol nlysis Liver lipis were extrte with hloroform-methnol (.9 g of tissue) oring to the metho esrie y Folh [3]. Cholesterol n triylglyerol in serum n liver were mesure with n enzymti olorimetri ommeril kit (DiSys Dignosti Systems, Holzheim, Germny) in hemistry nlyser (RA-5; Tehnion Ames, Trrytown, NY, USA). RNA isoltion n quntittive PCR The totl RNA from liver n ileum ws extrte s esrie y Chomzynski n Shi [3]. Totl RNA ws reverse-trnsrie n PCR mplifition performe (Applie Biosystems, Foster

3 Dietologi () 55: City, CA, USA) using TqMn ssys (Applie Biosystems). Assys for eh gene were rrie out in triplite in 96-well optil pltes with sequene etetion system (ABI Prism 7; Perkin-Elmer Applie Biosystems, Foster City, CA, USA). β-atin ws use s the invrint ontrol for liver n intestine nlyses. Histologil nlysis Liver setions were otine, fixe y immersion in % formlehye (vol/vol) issolve in phosphte uffer n susequently ehyrte n emee in prffin. Setions (3 μm with) were otine n stine with hemtoxylin n eosin. Ftty i nlysis Totl lipis from the liver were extrte s esrie y Folh [3] n the ftty is then methylte s previously esrie [33]. The methylte ftty is were nlyse y gs hromtogrphy (Agilent 685; Agilent, Snt Clr, CA, USA) with flme ionistion etetor, (Agilent)using n HP- pillry olumn (J&W Sientifi, Alny, CA, USA). Bile i mesurements The mounts of ile i were etermine from the gll ler (~.5 ml ile per niml) n fees. For mesurement of fel ile i exretion, stools from WT n Lxrα / mie were ollete uring the finl 3 ys of the stuy, n rie, weighe n groun. The ile i ws erivtise s esrie y Keller n Jhreis [34]. The trimethylsilyl ile is were nlyse y gs hromtogrphy (Agilent 685 with flme ionistion etetor) using pillry olumn (Innowx; J&W Sientifi) s previously esrie [34]. Cell ulture n o-trnsfetions HepG ells were grown in DMEM, with gluose (5 mmol/l), % fetl ovine serum, peniillin ( IU/ml) n streptomyin ( mg/ml), in humiifie CO inutor t 37 C. Cells were o-trnsfete with the empty expression vetor or n expression vetor ontining Lxrα or Lxrβ, long with reporter vetor ontining three repets of the onsensus LXRE lone in pgl3 Bsi (Promeg, Mison, WI, USA). Cells were seee in 4-well pltes, o-trnsfete for 8 h n genistein or izein e t the onentrtions inite. The syntheti lign, GW3965 (Enzo Life Sienes, Frmingle, NY, USA), n the nturl oxysterol, (R)-hyroxyholesterol (Sigm- Alrih, St. Louis, MO, USA), were use s positive ontrols. After 6 h of inution, the ells were ollete n lyse. The luiferse tivity ws mesure using ommeril luiferse ssy kit (luiferin-atp; BioThem, Umeå, Sween) n luminometer (Infinite ; Ten, Sn Jose, CA, USA). Culture of primry mouse heptoytes n trnsfetion Mouse heptoytes were isolte y the ollgense perfusion tehnique n seprte from non-prenhyml liver ells y entrifugtion t 35 g [35]. On y, primry heptoytes were plte in six-well plte (9.6 m /well) (Corning CellBIND, Tewksury, MA, USA). On y, mouse Lxrα or Lxrβ (lso known s Nrh) expression vetors (4 ng) were trnsfete using trnsfetion regent (FuE HD; Rohe Dignostis, Mnnheim, Germny). At 4 h fter trnsfetion, genistein or izein (5 μmol/l), n/or μmol/l T937 were e. Totl RNA from the heptoytes ws otine using Trizol regent. Protein extrtion n western lotting Primry mouse heptoytes were homogenise in lysis protein RIPA uffer ontining mmol/l soium fluorie, mmol/l soium orthovnte n omplete protese inhiitor oktil tlets (Rohe Applie Siene, Mnnheim, Germny). Totl protein (3 μg) ws loe on 8% polyrylmie gels, seprte y SDS-PAGE n trnsferre to polyvinyliene ifluorie (PVDF) memrne. Blots were loke with nonft ry milk (Bio-R, Herules, CA, USA) n inute overnight t 4 C with the following primry ntioies: nti etyl-coa roxylse (ACC) n nti phospho-acc t Ser79 (pacc) (Millipore, Temeul, CA, USA), n ntitin (Snt Cruz Biotehnology, Snt Cruz, CA, USA). The ns were nlyse using ImgeJ.4p igitl imging proessing softwre ( ). OGTT n intrperitonel insulin tolerne tests Mie were fste for 6 h efore the OGTT n insulin tolerne test (ITT). The OGTT ws performe y ministering gluose (. g/kg oy weight) y gvge. Insulin (.8 unit/kg) ws injete intrperitonelly. Bloo smples were otine vi til nik n gluose ws mesure with gluometer (OneTouh Ultr Au-Chek Sensor; Rohe Dignostis). The AUC vlues were lulte s follows: [(gl)t (gl)t)*(t T)]/ [36], where gl is gluose n T is time. Sttistil nlysis The results re presente s the mens ± SEM. The sttistil nlysis ws performe using one-wy ANOVA followe y Fisher s protete lest-squre ifferene test to etermine signifint ifferenes etween the groups. Differenes were onsiere signifint t p<.5. Anlysis ws y Sttview sttistil nlysis progrm, version 4.5 (Aus Conepts, Berkeley, CA, USA). Results Effet of SP on oy weight n serum lipis No ifferene in oy weight ws oserve etween WT n Lxrα / mouse groups fter 8 ys (ESM Tle ). However, the

4 47 Dietologi () 55: liver weight of Lxrα / mie fe iets with holesterol ws signifintly higher thn tht of the other groups. Serum holesterol levels in ll groups were omprle. The ition of holesterol to the iets signifintly inrese serum holesterol levels in Lxrα /, ut not in WT mie (Fig. ). The profile of serum holesterol prtiles in Lxrα / mie showe signifint inrese in the numer of therogeni prtiles in omprison with WT mie. In prtiulr, WT mie fe SP h the lowest mount of therogeni prtiles n the highest onentrtion of HDL prtiles (Fig. ). Serum triylglyerol i not inrese in Lxrα / mie on either n SP or sein iet (Fig. ). Regultion of hepti lipis y SP Mrosopilly, livers of WT mie fe sein (ESM Fig. ) were ple ompre with those fe SP (ESM Fig. ), this ifferene eing ugmente in Lxrα / mie (ESM Fig. e, f). The ition of holesterol to the SP or sein iets inrese the ftty pperne (ESM Fig., ), prtiulrly in Lxrα / mie (ESM Fig. g, h). The livers of WT mie fe high-holesterol iets showe inrese umultion of holesterol n triylglyerol ompre with WT mie on iets without holesterol, lthough those fe SP h lower lipi levels (ESM Fig. i, j). The livers of Lxrα / mie fe either SP or sein ontine signifintly higher onentrtions of oth lipis thn the livers of orresponing WT mie. Dietry holesterol rmtilly inrese the hepti onentrtion of holesterol n triylglyerol in Lxrα / mie (ESM Fig. i, j). Lxrα / mie fe sein iet exhiite.- n 3.3-fol inrese levels of hepti holesterol n triylglyerol, respetively, ompre with those fe iet without holesterol. In Lxrα / mie fe n SP iet with holesterol, the orresponing inreses were 7.3- n.8-fol, respetively, ompre with those on iets without holesterol. Effet of SP on hepti histologil normlities WT mie fe sein h some heptoytes with smll- n meiumsize ytoplsmi lipi vesiles (ESM Fig. ), with ition of holesterol inresing the numer of lrge lipi vesiles (ESM Fig. ). These histologil hnges were lerly smller in WT mie fe the SP iet (ESM Fig., ). In ontrst, heptoytes of Lxrα / mie fe sein only showe lrge lipi vesiles (ESM Fig. e); the ition of holesterol to the iet proue greter inrese in the numer of lrge lipi vesiles, resulting in severe hepti stetosis, n unnt hroni inflmmtory infiltrte n nerosis (ESM Fig. g). These normlities were ssoite with rmti inrese (5.-fol) in the serum levels of lnine trnsminse (ALT) ompre with Lxrα / mie fe the sein iet without holesterol (ESM Fig. k). Although these histologil hnges were lerly ttenute in Lxrα / mie fe SP or SP with holesterol (ESM Fig. f, h), the ition of holesterol inrese serum ALT levels y 4.9-fol (ESM Fig. k). Regultion y SP of hepti genes involve in ile i synthesis n RCT On mesuring the expression of genes tht re responsile for the synthesis of ile is n epenent on LXR, we oserve signifint ifferenes in the Cyp7 mrna onentrtion etween Lxrα / mie n WT mie fe either the sein or SP iets (Fig. ). Wheres only the expression of Cyp7 ws inrese in Lxrα / mie fe iets without holesterol, the ition of holesterol represse Cyp7 expression in WT n Lxrα / mie (Fig. ). Serum holesterol (mmol/l) Cholesterol (mmol/l) Serum triylglyerol (mmol/l).5.5,, Cs Soy Cs Soy Cs Soy Cs Soy - Chol % Chol - Chol % Chol Lxrα / Lxrα -/ Density (mg/ml) Cs Soy Cs Soy Cs Soy Cs Soy - Chol % Chol - Chol % Chol Lxrα / Lxrα -/- Fig. Serum lipi onentrtions in WT n Lxrα / mie fe soy or sein (Cs) iets with or without holesterol (Chol). () Serum holesterol onentrtions n () the lipoprotein profile of holesterol prtiles; lk imons, Soy Lxrα / ; lk tringles, Cs Lxrα / ; lk irles, SoyChol Lxrα / ; lk squres, CsChol Lxrα / ; white imons, Soy Lxrα / ; white tringles, Cs Lxrα / ;white irles, SoyChol Lxrα / ; white squres, CsChol Lxrα /.() Serum triylglyerol onentrtions. The mie were fe the experimentl iets for month n loo ws otine fter overnight fsting. The results re expresse s mens ± SEM; n mie per group; men vlues with ifferent supersript letters within row re signifintly ifferent (p<.5)

5 Dietologi () 55: Hepti Cyp7 mrn (Reltive expression) Totl ile i in gll ler (μg/μl of ile).5.5,, Hepti Cyp7 mrn (Reltive expression) Cs Soy Cs Soy Cs Soy Cs Soy Cs Soy Cs Soy Cs Soy Cs Soy - Chol % Chol - Chol % Chol - Chol % Chol - Chol % Chol Fel totl ile is (μg/g).5 4 e f e Soy Cs Soy Cs Soy Cs Soy Cs Soy Cs Soy Cs Soy Cs Soy - Chol % Chol - Chol % Chol - Chol % Chol - Chol % Chol We oserve signifint hnges in the expression of holesterol trnsporters. The ition of holesterol to the iet in WT n Lxrα / mie signifintly inrese the expression of the ATP-ining ssette (ABC) su-fmily genes A, Ag5 n Ag8, with groups fe SP hving the highest respetive mrna levels in the liver (ESM Fig. 3 ). The expression of these trnsporters in Lxrα / mie is possily meite y LXRβ. Anlysis of the reltive expression of these genes, se on the unne of hepti holesterol n lulte s the rtio of the reltive mrna unne n the hepti holesterol onentrtion, showe tht Lxrα / mie fe SP or sein h rmtilly erese expression upon the ition of ietry holesterol in omprison with WT mie, initing in the former n inility to remove holesterol (ESM Fig. 3 f). There were no ifferenes in the totl mount of ile is in the ile (Fig. ). However, fel totl ile is exrete from WT mie on n SP iet with holesterol were ninefol higher thn in the ontrol groups or in mie fe sein with holesterol (Fig. ). These ifferenes were not oserve in the Lxrα / groups. SP regultes ile i exretion n intestinl expression of RCT genes To unerstn this hnge in ile i exretion, we mesure the unne of holesterol n ile Lxrα / Lxrα -/- Lxrα / Lxrα -/ Lxrα / Lxrα -/- Lxrα / Lxrα -/- Fig. Hepti expression of genes enoing the enzymes of ile i metolism, n ile i onentrtion in WT n Lxrα / mie fe sein or SP iets with or without holesterol (Chol). () Cyp7 n () Cyp7 mrna expression. () Totl ile i onentrtion in ile n () totl fel ile is. The results re expresse s mens ± SEM; n mie per group; men vlues with ifferent supersript letters within row re signifintly ifferent (p<.5), i trnsporters in the ileum. The expression of Fxr (lso known s Nrh4), Ip (lso known s Fp6) n It (lso known s Sl) erese in WT mie fe highholesterol iets (ESM Fig. 4 ), wheres Lxrα / mie fe iets with or without holesterol i not express these genes t levels ove ontrol levels. Interestingly, WT mie fe SP without holesterol showe inrese Ag5, Ag8 n A mrna levels ompre with those fe sein (ESM Fig. 4 f). Moreover, the ition of holesterol inrese the expression of these genes y %, 3% n 4%, respetively, ompre with expression in mie fe sein. These ifferenes were olishe when Lxrα / mie were fe the experimentl iets with or without holesterol. Regultion of LXR isoform tivity y SP isoflvones To explore whether soy isoflvones were responsile for tivting proution of the ABC trnsporters vi LXRα or LXRβ, we onute funtionl ssys in HepG ells to nlyse the effet of the soy isoflvones (genistein n izein) on the LXREs. The ition of izein or genistein i not inrese luiferse tivity ompre with the respetive ontrols, wheres the syntheti lign, GW3965, n the nturl oxysterol, (R)-hyroxyholesterol, signifintly inrese luiferse tivity (ESM Fig. 5, ). These t strongly suggest tht the oserve inrese in the expression of genes enoing the ABC trnsporters ws not meite vi iret LXR gonist effet. Stuies with LXRβ showe similr results (t not shown). Next, we stuie whether isoflvones oul regulte LXRα iniretly. We showe tht the inution of heptoytes with T937 inrese Srep (lso known s Sref) mrna unne y 8.4-fol. Interestingly, the heptoytes trnsfete with the Lxrα vetor inute with izein or genistein signifintly reue the stimultory effet of T937 y 4% n 5%, respetively (Fig. 3). However, the repressive effet of isoflvones on Srep expression ws not oserve in heptoytes trnsfete with the Lxrβ vetor (Fig. 3). Similr effets were oserve for A, nother LXR trget gene (Fig. 3 ). Surprisingly, the response of the Ag5 n Ag8 genes ws the opposite of tht oserve for Srep n A. The inution of stimulte trnsfete heptoytes with the Lxrβ vetor in the presene of isoflvones further stimulte the mrna expression of Ag5 n Ag8, prtiulrly upon the ition of genistein (.4- to.8-fol) (Fig. 4 ). These t suggest tht isoflvones my ifferentilly regulte the expression of LXR trget genes. There is eviene tht the tivity of LXRα n e ownregulte y phosphoryltion of AMP-tivte protein kinse (AMPK) [5]. We therefore use the AMPK inhiitor, ompoun C, to explore whether isoflvones oul regulte the tivity of LXRα vi AMPK. As oserve in Fig. 5, the ility of T937 to inrese Srep

6 474 Dietologi () 55: mrna Srep reltive expression mrna A reltive expression Control T9 T9 T9 Control Control T9 T9 T9 T9 T9 Control T9 T9 T9 T9 Fig. 3 The effet of soy isoflvones on the expression of Srep n A in mouse heptoytes overexpressing Lxrα or Lxrβ. The ells were trnsfete with the expression vetor for (, ) Lxrα n (, )or Lxrβ, n the heptoytes were inute with izein () or genistein (), with or without the syntheti LXR lign T937 (T9). The experiments were performe in quruplite; men vlues with ifferent supersript letters within row re signifintly ifferent (p<.5) expression y tivting LXRα ws highly stimulte y the ition of ompoun C. Nonetheless, genistein ws le to repress the synergisti effet of T937, even in the presene of ompoun C. Conversely, in heptoytes overprouing LXRβ, genistein signifintly inrese the expression of Ag5 in the presene of T937 with ompoun C (Fig. 5). We showe tht genistein ws le to tivte AMPK, sine this isoflvone inrese the phosphoryltion of ACC, well-known trget protein of AMPK. The ition of T937 i not lter ACC phosphoryltion, while ompoun C, n inhiitor of AMPK, prevente the stimultory effet of genistein on ACC phosphoryltion (ESM Fig. 6, ). Regultion y SP of expression of genes involve in holesterol n ftty i synthesis We mesure the mrna levels of severl genes involve in hepti holesterol n ftty i metolism in WT n Lxrα / mie. Our t show tht the expression of Srep (lso known s Sref) n HMG-CoA reutse in the livers of WT mie ws signifintly reue in nimls fe holesterol iets (ESM Fig. 7, e); however, mrna levels for the LDL reeptor were not signifintly ifferent mong the WT groups (ESM Fig. 7f). Interestingly, Lxrα / mie fe sein or SP mrna Srep reltive expression mrna A reltive expression iets without holesterol h signifintly higher expression of Srep mrna n its trget genes, whih enoe HMG-CoA reutse n the LDL reeptor, thn the orresponing WT mie (ESM Fig. 7, e). However, levels of Srep mrna hnge only slightly mong WT groups (ESM Fig. 7). Agin, the expression of Srep n its trget gene Fsn were signifintly inrese in the liver of Lxrα / mie fe iets without holesterol, n effet tht ws represse y the ition of holesterol (ESM Fig. 7). Hepti S showe ifferent pttern of expression in WT n Lxrα / mie (ESM Fig. 7). Nonetheless, there ws no signifint orreltion etween S mrna n the sturte:monounsturte ftty i rtio in the liver. This rtio ws epenent upon the ition of holesterol to the iets (Tle ). WT or Lxrα / mie fe iets with holesterol h lower rtios thn the orresponing groups fe iets without holesterol. The rtios were etermine mostly y the high onentrtion of olete in the livers of mie fe high-holesterol iets n were inepenent of the type of ietry protein (Tle ). SP regultes insulin sensitivity There is strong eviene tht the umultion of lipis in musle n liver is ssoite mrna Ag5 reltive expression mrna Ag8 reltive expression , Control T9 T9 Control T9 T9 T9 T9 mrna Ag5 reltive expression mrna Ag8 reltive expression e e Control T9 T9 Control T9 T9 T9 T9 Fig. 4 The effet of soy isoflvones on the expression of Ag5 n Ag8 in mouse heptoytes overexpressing Lxrα or Lxrβ. Cells were trnsfete with the expression vetor for (, ) Lxrα or (, ) Lxrβ, n heptoytes were inute with izein () or genistein (), with or without the syntheti LXR lign T937 (T9). The experiments were performe in quruplite; men vlues with ifferent supersript letters within row re signifintly ifferent (p<.5)

7 Dietologi () 55: mrna Srep reltive expression Control T9 T9 CC T9 CC T9 CC mrna Ag5 reltive expression 5 e e e Control T9 T9 CC T9 CC T9 CC Fig. 5 () The effet of genistein on Srep expression in mouse heptoytes overexpressing Lxrα n inute with the syntheti LXR lign, T937 (T9), n/or with ompoun C (CC), n inhiitor of AMPK. () The effet of genistein on the expression of Ag5 in mouse heptoytes overexpressing Lxrβ; inution s ove (). The experiments were performe in quruplite; men vlues with ifferent supersript letters within row re signifintly ifferent (p<.5) with the evelopment of insulin resistne [37]. Consiering the elevte mounts of lipis in the livers of Lxrα / mie fe sein or SP high-holesterol iets, we investigte whether the type of ietry protein oul ifferentilly moify insulin sensitivity s mesure y the OGTT n ITT. Our results showe tht WT mie fe n SP high-holesterol iet h lower fsting loo gluose levels thn mie fe sein high-holesterol iet (Fig. 6). Serum gluose ispperne following n orl gluose lo in WT mie fe SP ws signifintly improve ompre with mie fe sein (Fig. 6, ). In ition, the ITT showe tht gluose ispperne from the loo ws improve y SP-fe mie ompre with sein-fe mie (Fig. 6) These t show tht WT mie fe SP h etter insulin sensitivity thn the orresponing mie on sein iet (Fig. 6). However, in Lxrα / mie fe sein or SP high-holesterol iets, no signifint ifferenes were oserve uring the OGTT or ITT (Fig. 6, ).Lxrα / mie h lower sl serum gluose onentrtion n improve insulin response ompre with WT mie fe sein high-holesterol iet, suggesting tht the presene of LXRα my prtilly ffet insulin sensitivity in mie fe sein (Fig. 6). Disussion The present stuy emonstrtes tht the onsumption of n SP iet with holesterol reues the umultion of hepti lipis even in Lxrα / mie. Mrosopi exmintion of the livers of WT mie fe SP holesterol iets showe tht Tle Conentrtion of sturte n monounsturte ftty is in the liver of WT n Lxrα / mie fe sein or SP iets with or without holesterol for 8 ys Wil-type Lxrα / Vrile Cs Soy CsChol SoyChol Cs Soy CsChol SoyChol Plmitte (μg/g tissue) 79±3, 4±43,, 49±33, 39±36, 8±9 35±, 35± 96±6 Plmitolete (μg/g tissue) 7±6, 9±5 8±, 88± 5±4 6±4 39±5 6± Sterte (μg/g tissue) 48±6, 63± 5±8, 4±3 5±3, 5±4, 58±4, 63±5 Olete (μg/g tissue) 38±4, 355±8, 487±68 598±, ±7 57±35 98±78 985±79 C6/C6: 7.±.4 4.5±. 3.±.,e 3.±.,e 8.8±. 9.5±.6.3±.4.4±.5,e C8/C8:.±..±.3.±.,e.7±. e.5±.8.4±.6.6±.5 e.6±. e S:M.9±..7±.5.5±.3.4±.,.4±..±..3±..3±.5 Vlues re mens ± SEM. Men vlues with ifferent supersript letters within row re signifintly ifferent (p<.5) Cs, sein; Chol, holesterol; S:M, sturte:monounsturte ftty i rtio

8 476 Dietologi () 55: Fig. 6 OGTT n ITT results in WT n Lxrα / mie. () The time urve of gluose onentrtions in loo uring n OGTT in WT (lk irles) n Lxrα / (white irles) mie fe sein, or in WT (lk squres) n Lxrα / (white squres) mie fe SP highholesterol iets for month. () The AUC from OGTT etile in (). Blk rs, sein iet; white rs, SP iet. () Time urve of gluose onentrtions in loo uring n ITT in WT n Lxrα / mie s in () ove. () The AUC from the ITT. Blk rs, sein iet; white rs, SP iet. The results re expresse s the mens ± SEM; n 5 7 mie per group; *p<.5 for the ifferene etween the soy n sein groups they were less ftty n h etter onsisteny thn those of mie fe sein iet with holesterol. Moreover, the effet of SP ws lso oserve in the livers of Lxrα / mie, whih normlly hve ftty pperne n very soft texture, s previously reporte [8, 3]. In ft, livers of Lxrα / mie fe n SP iet showe erese in lrge lipi eposits n reue inflmmtory response. These t suggest tht SP hs n nti-stetoti effet, s oserve in previous stuies [3 5]. Previous stuies n the present work hve shown tht the elimintion of exess ietry holesterol to prevent its hepti umultion ours vi inrese fel ile i exretion [8]. We i not oserve n inrese in Cyp7 n Cyp7 expression in mie fe long-term highholesterol iets, nor i we see signifint ifferenes in the onentrtion of totl ile is in the ile. However, WT mie fe n SP iet with holesterol h inrese liver n intestinl expression of Ag5, Ag8 n A, n effet not oserve in Lxrα / mie. The highest levels of these trnsporters in WT mie were ssoite with the highest onentrtion of totl fel ile is. Although the expression of genes involve in ftty i synthesis, ile i synthesis n RCT is regulte y LXR [9], our t show tht onsumption of SP upregulte some of the genes involve in these proesses n ownregulte the expression of some others. SP isoflvones were le to meite the effets of LXR, sine they re ligns for other nuler reeptors, suh s the peroxisome prolifertor-tivte reeptors n estrogen reeptors [7 9, 38]. However, our t inite tht isoflvones re unle to work s iret LXR gonists n therey stimulte trnsription vi lssil LXRE, fining tht is in greement with previous results [39]. Our results in heptoytes suggest tht isoflvones re le to regulte LXR tivity iniretly y promoting the phosphoryltion, possily meite vi AMPK, of LXRα or LXRβ, leing to opposing effets on the expression of ertin genes. Isoflvones reue the expression of Srep n A vi LXRα, ut t the sme time inrese the expression of Ag5 n Ag8 vi LXRβ. Our t on the use of ompoun C, n inhiitor of AMPK, support the hypothesis tht isoflvones re le to tivte AMPK, leing to the repression of Srep y the phosphoryltion of LXRα n the overexpression of Ag5 y the phosphoryltion of LXRβ. Our group n others hve lso emonstrte tht isoflvones re le to inrese the phosphoryltion stte of AMPK [4, 4]. More stuies re neee to unerstn the full mehnism of this tivtion. The umultion of hepti lipis hs reently een esrie s one of the min uses for the evelopment of the metoli normlities oesity, primrily insulin resistne n yslipiemi [4]. Our results lerly show tht feeing n SP high-holesterol iet to WT mie improves insulin sensitivity ompre with WT mie on sein high-holesterol iet. These results re in greement with previous stuy initing tht SP improves insulin sensitivity [6]. However, this enefiil effet ws olishe following the eletion of Lxrα, initing tht the effet of SP on gluose metolism involves LXRα. Interestingly, Lxrα / mie h etter insulin sensitivity inepenently of the type of iet, suggesting tht the sene of LXRα in vivo hs enefiil rther thn negtive effet on insulin sensitivity. This is in greement with previous t from our group, whih showe tht Lxrα / mie re more insulin-sensitive thn WT mie, even fter high-ft iet leing to liver stetosis [36]. The enefiil effets of lk of LXRα our vi LXRβ, sine this isoform ts in the opposite iretion to LXRα in gluose metolism n insulin sensitivity [36]; it is lso the min isoform in musle tissue [43] n is present in ipoytes, leing to n improvement of whole-oy insulin sensitivity. This suggests tht the improvement of insulin sensitivity y n SP iet oul e meite y LXRα. In support of this fining, Lxrβ / mie fe the SP iet showe improve insulin sensitivity (t not shown).

9 Dietologi () 55: In onlusion, the ifferentil expression of genes regulte y LXR fter the onsumption of SP is in prt ue to the pity of isoflvones, prtiulrly genistein, to regulte the tivity of the LXR isoforms, LXRα n LXRβ, in opposing iretions, possily meite vi AMPK. The ifferent effets of isoflvones on these nuler reeptors re likely to explin the enefiil results oserve in vrious stuies of experimentl nimls n in humns, where the onsumption of n SP iet ws shown to reue serum holesterol n prevent the exessive umultion of hepti lipis, s well s improving insulin sensitivity [3 5, 44, 45]. Further stuies re neee to unerstn the moleulr mehnisms of the regultion of LXR isoforms y isoflvones. Funing This work ws supporte y the Roert A. Welh Fountion, CONACYT Mexio (grnts to O. Grnos n 4635-M to N. Torres) n the Sweish Reserh Counil (ontrt numer to K.R. Steffensen; ontrt numer to J.A. Gustfsson). M. González-Grnillo reeive sholrship from CONACYT. 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