Naringenin prevents cholesterol-induced systemic inflammation, metabolic dysregulation and atherosclerosis in Ldlr -/- mice.

Transcription

1 Nringenin prevents cholesterol-induced systemic inflmmtion, metolic dysregultion nd therosclerosis in Ldlr -/- mice. Juli M. Assini 1,2, Erin E. Mulvihill 1,2, Brin G. Sutherlnd 1, Dwn E. Telford 1,3, Cynthi G Swyez 1,3, Srh L. Felder 1,2, Snjiv Chhoker 1,2, Jne Y. Edwrds 1,3, Roert Gros 1,3,4 nd Murry W. Huff 1,2,3, * 1 Vsculr Biology, Rorts Reserch Institute, 1 Perth Drive, London, Ontrio, Cnd, N6A 5K8. 2 Deprtment of Biochemistry, 3 Deprtment of Medicine, nd 4 Deprtment of Physiology nd Phrmcology, The University of Western Ontrio, London, Ontrio, Cnd, N6A 5C1. *Correspondence: mhuff@uwo.c Murry W. Huff Rorts Reserch Institute, Perth Drive, London, Ontrio, Cnd, N6A 5K8 Phone: +1 (519) FAX: +1 (519) Running Title: Nringenin prevents cholesterol-induced inflmmtion ABBREVIATIONS: ABCG, ATP-inding cssette su-fmily G; ACOX, Acetyl-CoA oxidse; AP-1, ctivted protein 1; AUC, re under the curve; CE, cholesteryl ester; CPT-1α, crnitine plmitoyl trnsferse-1 lph; CYP7A1, cholesterol 7lph-hydroxylse; FA, ftty cid; FC, free cholesterol; FGF21, firolst growth fctor 21; FPLC, fst performnce liquid chromtogrphy; GTT, glucose tolernce test; HFHC, high-ft, high-cholesterol; HMGCR, 3-hydroxy-3-methyl-glutryl-CoA reductse; IHC, immunohistochemistry; IL, interleukin; LF, low-ft; LFHC, low-ft, highcholesterol; LXR, liver X receptor; MAPK, mitogen-ctivted protein kinse; MCP-1, CCL2, chemokine (C-C motif) lignd 2; CCL3, chemokine (C-C motif) lignd 3; MOMA-2, monocyte mcrophge ntiody 2; NF-κB, nucler fctor kpp B; PGC-1α, peroxisome prolifertorctivted receptor gmm coctivtor 1-lph; SAA, serum myloid ; SM α-ctin, smooth muscle lph ctin; SREBF, sterol regultory element-inding fctor; TC, totl cholesterol; TG, triglyceride; TNFα, tumor necrosis fctor lph; WAT, white dipose tissue. Downloded from y guest, on June 19, 218 1

2 Astrct: Oesity-ssocited chronic inflmmtion contriutes to metolic dysfunction nd propgtes therosclerosis. Recent evidence suggests tht incresed dietry cholesterol excertes inflmmtion in dipose tissue nd liver, contriuting to the pro-therogenic milieu. The ility of the citrus flvonoid nringenin to prevent these cholesterol-induced perturtions is unknown. To ssess the ility of nringenin to prevent the mplified inflmmtory response nd therosclerosis induced y dietry cholesterol, mle Ldlr -/- mice were fed either cholesterolenriched high-ft or low-ft diet supplemented with 3% nringenin for twelve weeks. Nringenin, through induction of heptic ftty cid (FA) oxidtion nd ttenution of FA synthesis, prevented heptic stetosis, heptic VLDL overproduction nd hyperlipidemi induced y oth cholesterolrich diets. Nringenin ttenuted heptic mcrophge infiltrtion nd inflmmtion stimulted y dietry cholesterol. Insulin resistnce, dipose tissue expnsion nd inflmmtion were llevited y nringenin. Nringenin ttenuted the cholesterol-induced formtion of oth fom cells nd expression of inflmmtory mrkers in peritonel mcrophges. Nringenin significntly decresed therosclerosis nd inhiited the formtion of complex lesions, which ws ssocited with normlized ortic lipids nd reversl of ortic inflmmtion. We Downloded from y guest, on June 19, 218 demonstrte tht in mice fed cholesterol-enriched diets, nringenin ttenutes peripherl nd systemic inflmmtion, leding to protection from therosclerosis. These studies offer therpeuticlly relevnt lterntive for the prevention of cholesterol-induced metolic dysregultion. Astrct Word Count: 23 Supplementry Key Words: Inflmmtion, Atherosclerosis, Cholesterol, Flvonoid, Lipids, Oesity, Heptic Stetosis, Insulin Resistnce, Metolism. 2

3 Introduction Metolic syndrome is collection of normlities including oesity, dyslipidemi, hypertension, nd insulin resistnce, ll of which contriute to the development of type 2 dietes nd therosclerosis. Insulin resistnce, dyslipidemi nd therosclerosis re mplified y the development of chronic low-grde inflmmtory response (1). In insulin resistnt sttes, monocyte-derived mcrophges infiltrte viscerl dipose tissue, resulting in pro-inflmmtory cytokine synthesis, either from dipocytes or resident mcrophges, which impirs insulin sensitivity (2,3). Administrtion of diets rich in sturted fts to Ldlr -/- mice represents model with mny chrcteristics of the metolic syndrome (4-6). Recent studies in this model hve implicted elevted dietry cholesterol in the induction of mcrophge infiltrtion nd the inflmmtory response in oth dipose tissue nd liver, leding to the excertion of dyslipidemi, insulin resistnce, nd therosclerosis (6,7). Cholesterol-fed Ldlr -/- mice disply incresed circulting serum myloid A (SAA), n inflmmtory meditor tht potentites inflmmtion in the rtery wll (8). In hmsters, dietry cholesterol functions synergisticlly with dietry ft nd fructose to intensify dyslipidemi, insulin resistnce nd heptic stetosis (9). Other studies estlished link etween heptic Downloded from y guest, on June 19, 218 inflmmtion nd dyslipidemi; tumor necrosis fctor α (TNFα)- nd interleukin (IL)-1β stimulted heptic overproduction of pob1-contining lipoproteins in vivo nd in vitro (1,11). Collectively, these studies indicte tht moderte increse in dietry cholesterol promotes tissue nd systemic inflmmtion, therey contriuting to dyslipidemi, insulin resistnce nd therosclerosis. Phrmcologicl therpies for the tretment nd prevention of the chronic low-grde inflmmtion ssocited with therosclerosis remin elusive. Epidemiologicl studies reveled n ssocition etween incresed consumption of dietry flvonoids nd reduced risk of crdiovsculr disese (12). Flvonoids hve een 3

4 shown to hve nti-inflmmtory properties (13). Nringenin, citrus flvonoid inhiits pob1 secretion from cultured heptocytes (14). Furthermore, in Ldlr -/- mice, ddition of nringenin to high-ft diet ttenutes oesity nd prevents heptic triglyceride (TG) ccumultion through incresed heptic ftty cid (FA) oxidtion, leding to prevention of dyslipidemi, insulin resistnce nd therosclerosis (5,15). These studies were performed in the context of diets low in cholesterol (.5%). Due to the evidence tht high dietry cholesterol ugments the inflmmtory stte nd potentites therogenesis (1), one ojective of the present study ws to exmine the ility of nringenin to prevent these cholesterol-induced perturtions. Diets low in ft, ut high in sucrose nd contining moderte mounts of cholesterol (.2%) induce significnt therosclerosis in Ldlr -/- mice (16). However, the effect of dietry cholesterol in low ft diets, on the locl nd systemic inflmmtory response hs not een determined. Furthermore, the ility of nringenin to prevent metolic dysfunction nd therosclerosis in the context of low-ft, therogenic diet, remins to e elucidted. Therefore, second ojective ws to exmine the ility of nringenin to prevent inflmmtion, metolic dysregultion nd therosclerosis in mice fed low-ft (LF), cholesterol-contining diet. In the current study, in Ldlr -/- mice fed either high-ft, high-cholesterol (.2%) (HFHC) Downloded from y guest, on June 19, 218 diet or low-ft, high-cholesterol (.2%) (LFHC) diet, nringenin supplementtion prevented dyslipidemi nd hyperinsulinemi. By enhncing heptic FA oxidtion nd ttenuting lipogenesis, nringenin prevented heptic stetosis. The cholesterol-induced inflmmtion in liver, dipose tissue, nd ort were llevited y nringenin. Collectively, the correction of metolic normlities nd the inflmmtory response ws ssocited with mrked ttenution of therosclerosis. 4

5 Methods Animls nd diets Mle Ldlr -/- mice, 8-12 weeks of ge, on C57BL/6J ckground were otined from Jckson Lortories (Br Hrour, ME). Mice were fed d liitum for 12 weeks (n= 1-12/group) either stndrd chow diet (14% kcl ft, TD864, Hrln Tekld, Mdison, WI), high-ft, high-cholesterol diet (HFHC, 42% kcl ft,.2% cholesterol, TD9268, Hrlnd Tekld) ± 3% wt/wt nringenin (Sigm-Aldrich, St. Louis, MO), low-ft, semi-synthetic diet (LF, 12.4% kcl ft, 52% kcl sucrose, AIN-76A, Hrln Tekld), or low-ft, high-cholesterol diet (LFHC, 12.4% kcl ft, 52% kcl sucrose,.2% cholesterol, TD981, Hrln Tekld) ± 3% wt/wt nringenin. Complete dietry composition is listed in Supplementry Tle I. All experiments were hndled in ccordnce with the Cndin Guide for the Cre nd Use of Lortory Animls nd pproved y the University of Western Ontrio Animl Cre Committee. Blood nd tissue collection Mice were fsted for 6 h efore scrifice. Blood nd tissue collection were performed s descried previously (5,15,17). Animls were nesthetized with Avertin, lood ws collected vi crdic puncture nd stored t -2 C. Herts were perfused with heprin-pbs nd the hert Downloded from y guest, on June 19, 218 nd full-length ort were dissected together. The top hlf of the hert ws removed, plced in Optimum Cutting Temperture (OCT) medium nd frozen on dry ice. The ort ws plced in 1% formlin. Other tissues including liver, muscle nd dipose tissue were removed, weighed, snp frozen in liquid nitrogen nd stored t -8 C. Plsm nd tissue nlysis Plsm concentrtions of insulin, leptin (ALPCO Dignostics, Windhm, NH, USA) nd SAA (Invitrogen, Life Technologies, Mississug, ON) were determined y mouse-specific ELISA s per mnufcturer s instructions. Plsm concentrtions of TG nd totl cholesterol 5

6 (TC) were mesured enzymticlly (5). The TC nd TG within VLDL, LDL nd HDL prticles were determined in fresh plsm y fst performnce liquid chromtogrphy (FPLC) s descried previously (5). Gll ldder ile cids were mesured using n enzymtic ssy (18). Lipids were extrcted from whole ort nd 1 mg of liver using the method of Folch et l.(19) nd quntitted s descried previously (2). FA nd cholesterol synthesis were mesured following i.p injection of [1-14 C]-cetic cid (5). FA oxidtion ws determined in tissue homogentes y conversion of [ 3 H]-plmitte to 3 H 2 O (5). Glucose nd insulin tolernce tests Glucose Tolernce Tests (GTT) were conducted in mice fsted for 6 h nd injected i.p with 15% D-glucose solution in.9% NCl (1 g/kg ody weight). Blood glucose ws mesured using glucometer (Acensci Elite, Byer Helthcre, Toronto, ON) t regulr intervls up to 12 min post-injection. Insulin Tolernce Tests (ITT) were conducted fter 5 h fst nd i.p. injection with.6 IU/kg Novolin ge Toronto (Novo Nordisk, Mississug, ON). Blood glucose ws mesured s descried ove every 15 min for 1 h. Glucose utiliztion nd insulin sensitivity were clculted sed on the solute re under the curve (AUC). Triglyceride nd pob1 secretion; triglyceride nd cholesterol sorption Downloded from y guest, on June 19, 218 TG nd pob1 secretion into plsm ws determined following i.p. injection of tyloxpol nd 2 μci of Trn [ 35 S]-lel (1 Ci/mmol, MP Biomedicls Inc., Irvine, CA) (for pob1 secretion only) (5). Mice were scrificed y CO 2 inhltion nd lood ws collected vi crdic puncture. A comined VLDL/IDL frction (density <1.19 g/ml) ws isolted from 25 μl of fresh plsm y ultrcentrifugtion nd seprted on 4.5% SDS-PAGE gels (14). Intestinl cholesterol sorption ws determined using modified fecl isotope rtio method (5). Mice were gvged with 15 μl of medium chin triglyceride oil (Novrtis Medicl Nutrition, Fremont, MI) contining 1 μci of [4-14 C]-Cholesterol (Perkin Elmer, Wlthm, MA) or 2.5 µci of glycerol 6

7 tri[1-14 C]olete nd 2. μci of [5,6-3 H]-β-sitostnol (Americn Rdioleled Chemicls Inc., St. Louis, MO). Feces were collected every 24 h for three dys. Fecl smples were sponified in 95% ethnol/1n KOH, neutrl lipids were extrcted with hexne nd the percentge of lipid sored ws clculted s the rtio of [ 14 C] to [ 3 H] (5). Tissue sectioning nd histology Pieces of liver were plced in OCT, frozen nd sectioned t 8 μm using cryostt. Liver sections were stined with Oil Red O nd Hoechst (2.5 μg/ml, Sigm), nd visulized y fluorescence microscopy t 493 nm excittion. Immunohistochemistry (IHC) ws conducted on liver sections (frozen) for the mcrophge mrker MAC-2 (Cedrlne, Burlington, ON). Briefly, slides were fixed in cetone, quenched with 3% H 2 2 in methnol, locked in 1% BSA nd incuted with rt nti-mouse MAC-2 ntiody (1:1), followed y incution with secondry ntiody (iotinylted got nti-rt IgG ntiody, 1:2, Vector, Burlington, ON). Sections were incuted in ABC regent (Vector) followed y incution in DAB sustrte (Vector) nd counterstined in hemtoxylin. White dipose tissue (WAT) ws fixed in 1% formlin, emedded in prffin, sectioned using microtome nd stined with hemtoxylin nd eosin. Adipocyte dimeter ws clculted Downloded from y guest, on June 19, 218 using Northern Eclipse 7. softwre on 2x mgnifiction photomicrogrphs. Morphometric nlysis of lesions in the ortic sinus ws performed s descried previously (15). Frozen seril sections were stined with Oil Red O for quntittion of lesion re nd collgen ws visulized y stining slides with modified Verhoff nd Msson s Trichrome. Mcrophges were identified y IHC stining with monocyte/mcrophge ntiody-2 (MOMA-2, Accurte Chemicl nd Scientific Corportion, Westury, NY) nd smooth muscle cells identified y stining with SM α-ctin ntiody (Clone 1AH, Sigm-Aldrich). The reltive re of lesions positively stined for MOMA-2, collgen (trichrome) nd SM α-ctin ws 7

8 determined reltive to the totl re of the respective plque. Clened, full-length orts were fixed in formlin, stined with Sudn IV, prepred en fce nd mounted in glycerol geltin s descried previously (15). Lesion re ws clculted y dividing the stined plque re y the totl re of the ortic rch (scending nd descending ort) nd expressed s percentge. Gene expression nlysis Tissue mrna levels were determined y quntittive rel-time PCR s pulished previously (5). The expression of ech gene ws normlized to Gpdh expression nd the vlue of the control groups (Chow or LF) were set to 1. Energy expenditure Energy expenditure, respirtory quotient, nd totl ctivity were ssessed using the Comprehensive Lortory Animl Monitoring System (CLAMS) (Columus Instruments, Columus, OH). Len ody mss ws clculted from whole ody composition nlysis conducted y micro-ct imging using Locus Ultr micro-ct scnner (GE Helthcre, London, ON) (21). Peritonel mcrophges In seprte study, mle Ldlr -/- mice 8-12 weeks of ge (n= 16/group) were fed either Downloded from y guest, on June 19, 218 stndrd chow or the HFHC diet ± 3% (wt/wt) nringenin. After 12 weeks, mice were injected i.p. with 2.5 ml 4% Brewer s Thioglycollte Medium (Sigm-Aldrich), nd 5 dys lter mice were scrificed vi CO 2 inhltion. Peritonel cells were plted t density of x1 6 per well nd were llowed to dhere for 2 h (22). A suset of peritonel cells were plted into wells contining cover slips nd stined with Oil Red O. Cellulr TC, cholesteryl ester (CE) nd TG mss in peritonel cells were determined enzymticlly following extrction with 3:2 (vol:vol) hexne:isopropnol (23). Cellulr lipids were normlized to cell protein. Cellulr mrna levels were determined y quntittive rel-time PCR (5). 8

9 Sttisticl nlysis Dt is presented s the men ± SEM. A one-wy ANOVA nd post hoc Tukey test ws performed using Sigm Plot version 14. to determine sttisticl significnce. Different letters indicte sttisticl significnce within ech seprte study (P<.5); Chow, HFHC, nd HFHC + nringenin or LF, LFHC, nd LFHC + nringenin. A two-smple t-test ws lso employed to test the pre-specified hypothesis tht there is difference etween Chow nd LF groups (Asterisk (*) indictes sttisticl significnce) (P<.5). Downloded from y guest, on June 19, 218 9

10 Results Nringenin prevents dyslipidemi nd pob1 overproduction in cholesterol-fed mice Hypercholesterolemi ws induced in Ldlr -/- mice fed the HFHC nd LFHC diets for 12 weeks, wheres nringenin-supplementtion reduced plsm cholesterol y >4% (Tle 1). Plsm TG ws significntly incresed in mice fed HFHC (6-fold), LF (3-fold) nd LFHC (3-fold) compred to chow. Addition of nringenin to the HFHC or LFHC diets ttenuted plsm TG concentrtions y ~5% (Tle 1). FPLC reveled tht oth cholesterol-enriched diets mrkedly incresed VLDL- nd LDL-cholesterol, nd incresed VLDL-TG concentrtions (2- to 4-fold) (Fig. 1A nd 1B). In contrst, nringenin ttenuted VLDL- nd LDL-cholesterol levels, nd sustntilly reduced VLDL-TG, in the presence of either high or low dietry ft (Fig. 1A nd1b). Decresed plsm lipids y nringenin were independent of ny effect on cholesterol or TG sorption (Tle 1). The improvement in plsm lipids y nringenin ws primrily due to significnt reduction in heptic TG secretion into plsm (>55%) compred to HFHC- nd LFHC-fed nimls (Fig. 1C). Furthermore, HFHC- nd LFHC-fed mice secreted more pob1 (2.2- nd 1.6-fold, respectively) into plsm compred to chow-fed mice, which ws ttenuted (>8%) y nringenin (Fig. 1D). Downloded from y guest, on June 19, 218 Improved liver nd muscle lipid metolism y nringenin supplementtion to cholesterol-rich diets Prominent heptic stetosis developed in mice fed either cholesterol-rich diet, chrcterized y significntly elevted TC, CE, free cholesterol (FC) nd TG concentrtions compred to controls. Nringenin supplementtion of the HFHC or LFHC diet decresed heptic TC nd CE (>6%) nd normlized heptic TG (Fig. 2A nd 2B). Fluorescence photomicrogrphs of Oil Red O-stined heptic sections reveled sustntil reduction in neutrl lipid droplets in nringenin-supplemented, cholesterol-fed mice (Fig. 2C nd 2D). 1

11 Nringenin did not lter the cholesterol-induced suppression of sterol regultory element inding protein (Sref) 2 or 3-hydroxy-3-methyl-glutryl-CoA reductse (Hmgcr) expression (Fig. 2E nd 2F) or heptic cholesterol synthesis (Fig. 2G). Compred to HFHC or LFHC diets, nringenin significntly incresed cholesterol 7 lph-hydroxylse (Cyp71) expression (>5%) (Fig. 2E nd 2F), suggesting incresed conversion of cholesterol to ile cids. However, gll ldder ile cid content ws not ffected (Tle 1). Nringenin did not lter heptic expression of ATPinding cssette su-fmily G (Acg) 5, Acg8, or Ac1, lthough nringenin reduced Acg1 mrna compred to the HFHC or LFHC diets (Supplementry Figure I). The HFHC nd LFHC diets incresed heptic Sref1c expression (4- nd 1.8-fold, respectively), compred to controls, wheres nringenin significntly reduced Sref1c mrna (>3%) when dded to either cholesterol-rich diet (Fig. 2E nd 2F). De novo FA synthesis ws significntly incresed in cholesterol-fed nimls compred to controls, which ws rogted y nringenin (Fig. 2H). Compred to the HFHC or LFHC diets, nringenin incresed the expression of firolst growth fctor 21 (Fgf21) (3.6- nd 2-fold, respectively), peroxisome prolifertor-ctivted receptor gmm coctivtor 1 lph (Pgc1) (1.2- nd 1.3-fold), nd crnitine plmitoyl trnsferse-1 lph Cpt1 (1.8- nd 1.4-fold, respectively). Addition of Downloded from y guest, on June 19, 218 nringenin to the LFHC diet incresed cetyl-coa oxidse 1 (Acox1) mrna 2-fold, ut not in mice fed HFHC plus nringenin (Fig. 2E nd 2F). Heptic FA oxidtion ws significntly incresed in nringenin-supplemented mice compred to either cholesterol enriched diet lone (Fig. 2I). In muscle, nringenin normlized the incresed FA synthesis in HFHC, LF nd LFHC mice, ut did not impct muscle FA oxidtion (Supplementry Figure II). To evlute the incresed heptic FA oxidtion y the ddition of nringenin to either HFHC or LFHC diets, energy lnce ws ssessed in n niml metolic monitoring system. Totl ctivity ws similr mong ll dietry groups (Tle 1). Totl energy expenditure (EE) in 11

12 mice fed the HFHC diet ws lower (-14%) thn in chow-fed mice, which ws significntly incresed (16%) y the ddition of nringenin to the HFHC diet. Similrly, nringenin supplementtion of the LFHC diet significntly incresed EE (15%) compred to the LFHC diet lone (Fig 2J nd Supplementl Figure IIE). There ws no significnt chnge in cloric intke y the ddition of nringenin to either the HFHC or LFHC diets (Tle 1). Respirtory quotient (RQ) profiles, which reflect the reltive contriutions of crohydrte nd ft oxidtion to totl EE, were decresed in HFHC-fed mice reltive to chow, ut were not further ffected y the ddition of nringenin to the HFHC. Furthermore, RQ ws not chnged y the ddition of nringenin to the LFHC diet (Tle 1). Given the significnt increse in totl EE in nringenin-treted mice (Fig. 2J), the lck of chnge of RQ profiles suggest tht oth ft nd crohydrte oxidtion were incresed in the nringenin-treted mice. Nringenin prevents the development of cholesterol-induced heptic inflmmtion. In Ldlr -/- mice, incresed dietry cholesterol induces heptic inflmmtion (7,24). The HFHC diet significntly induced the expression of Tnf, Il1, chemokine (C-C motif) lignd 2 (Ccl2), chemokine (C-C motif) lignd 3 (Ccl3), tissue mcrophge specific mrker F4/8, nd serum myloid (S1/2) (Fig. 3A). Conversely, nringenin supplementtion to the HFHC diet Downloded from y guest, on June 19, 218 significntly ttenuted the expression of Tnfα, Il1, Ccl2, Ccl3 (>8%) nd reduced F4/8 nd S1/2 mrna (>5%) (Fig. 3A). Decresed expression of the mcrophge mrker F4/8 ws confirmed microscopiclly. Enhnced heptic mcrophge ccumultion (MAC-2) in HFHC-fed mice ws llevited y nringenin (Fig. 3C). The LFHC diet lso stimulted heptic inflmmtory gene expression (Tnf, Il1, Ccl2, Ccl3, nd S1/2) nd incresed MAC-2-stined cells (Fig. 3B nd 3D), lthough these increses were not s gret s in HFHC-mice. Nringenin-tretment normlized the expression of inflmmtory genes (Fig. 3B) nd mrkedly reduced the presence of MAC-2 positive cells 12

13 (Fig. 3D). Decresed heptic S1/2 expression in nringenin-treted mice ws ssocited with 7% reduction in plsm SAA1/2 concentrtions in oth dietry groups (Tle 1). Nringenin ttenutes dipose tissue expnsion nd inflmmtion nd restores glucose homeostsis in mice fed cholesterol-enriched diets In oesity, dipocytes expnd to ccommodte incresed TG nd re ssocited with incresed inflmmtion nd insulin resistnce (25). Dietry cholesterol my e stimulus for oth mcrophge infiltrtion into dipose tissue nd inflmmtory cytokine production (6). The HFHC diet incresed the diposity index 3-fold compred to chow-fed mice (Fig. 4A). Both diposity nd weight gin were completely normlized y ddition of nringenin to the HFHC diet (Fig. 4A nd Tle 1). Although the increse in diposity index in LF-fed mice ws not incresed further in LFHC-fed mice, supplementtion with nringenin reduced dipose tissue ccumultion to the levels of chow-fed mice (Fig. 4A nd Tle 1). The reduction in dipose tissue y the ddition of nringenin to either cholesterol-enriched diet ws independent of ny effect on cloric intke or TG sorption (Tle 1). The HFHC diet induced dipocyte hypertrophy (91-12 μm) compred to chow-fed mice (31-9 μm), wheres nringenin decresed dipocyte size (31-6 μm) (Fig. 4B). The LF diet Downloded from y guest, on June 19, 218 incresed dipocyte size (61-9 μm) compred to chow, nd ddition of cholesterol did not further increse dipocyte size (61-9 μm) (Fig. 4C). Nringenin tretment resulted in smller dipocytes (31-6 μm). Concomitnt with reduced dipose tissue expnsion y nringenin, plsm leptin concentrtions were normlized (Tle 1). Expnded dipose tissue in HFHC-fed mice ws ssocited with significntly incresed expression of inflmmtory genes including Tnf nd Ccl2, nd stimulted mcrophge infiltrtion s mesured y F4/8 mrna expression (Fig. 4D). Nringenin completely rogted the expression of these inflmmtory mrkers. Addition of cholesterol to the LF-diet mrkedly induced Tnf, Il1 nd Ccl3 expression, 13

14 demonstrting the impct of incresed dietry cholesterol lone on dipose tissue inflmmtion (Fig. 4E). Nringenin significntly decresed Il1, Il6, Ccl2 nd Ccl3 mrna compred to LFHCfed mice. F4/8 mrna ws unffected, suggesting tht dipose tissue mcrophge infiltrtion in LFHC-fed mice ws miniml compred to the LF diet lone (Fig. 4E). HFHC-fed mice were modestly hyperglycemic (1.3-fold) ut significntly hyperinsulinemic (3.8-fold) compred to chow-fed nimls (Tle 1). In contrst, nringenin completely normlized lood glucose nd plsm insulin levels. The LF diet incresed lood glucose (1.3-fold) nd plsm insulin (2.3-fold) compred to chow. The ddition of cholesterol to the LF diet did not further impct glucose or plsm insulin, however nringenin reduced oth prmeters to levels oserved in chow-fed mice (Tle 1). Nringenin normlized oth the impired glucose tolernce nd insulin sensitivity induced y the HFHC diet (Fig. 4F nd 4G). No difference ws oserved in either the glucose excursion or the response to exogenous insulin etween LF- nd LFHC-fed mice, wheres nringenin supplementtion significntly improved glucose nd insulin tolernce (Fig. 4F nd 4G). Nringenin prevents therosclerosis nd lters plque morphology in the ort of cholesterol-fed mice Downloded from y guest, on June 19, 218 We next evluted if nringenin s ility to ttenute plsm nd heptic lipids, improve glucose nd insulin homeostsis, nd prevent the inflmmtory response extended to protection from therosclerosis. Compred to chow, oth the HFHC nd LFHC diets mrkedly incresed Oil Red O-stined plque re in the ortic sinus to similr extent (~4.5 µm 2 x 1 5 ) (Fig. 5A nd 5B). Conversely, nringenin tretment prevented lesion development in the ortic sinus y ~5% when dded to either cholesterol-rich diet. Lesion re within the ortic rch ws sustntilly incresed in HFHC- nd LFHC-fed mice to 14% of surfce re (Fig. 5C nd 5D). 14

15 Addition of nringenin to either diet reduced plque re y >4%, which is depicted in representtive photogrphs of orte stined with Sudn IV (Supplementry Figure III). Anlysis of lesion morphology reveled tht in cholesterol-fed mice, the Oil Red O- stined lesion re ws ssocited with MOMA-2 stining, indicting the presence of lipid-rich, mcrophge fom cells (Supplementry Figures IV nd V). Plques in oth cholesterol-fed groups lso stined positively for collgen, which corresponded to the extent of SM α-ctinstined re, suggesting tht incresed smooth muscle cells within lesions contriuted to the secretion of collgen firils (Supplementry Figures IV nd V). Quntittively, the cholesterolrich diets induced lesions in which the percentge of stined lesion re occupied y MOMA-2 ws lower thn controls, with concomitnt increse in the percentge re occupied y collgen nd SM α-ctin (Fig. 5E nd 5F). These dt suggest tht cholesterol-fed mice develop complex lesions tht re not only rich in lipid ut lso in collgen, representing more firous phenotype. In contrst, in plques from nringenin-fed mice the percentge of MOMA-2- stined mcrophges ws elevted. These lesions contined less collgen nd SM α-ctin, indictive of lesions t n erly stge of development (Fig. 5E nd 5F, Supplementry Figures IV nd V). Thus, in ddition to reducing plque re, nringenin ttenutes the development of Downloded from y guest, on June 19, 218 complex lesions. Nringenin prevents lipid ccumultion nd inflmmtion in the ort nd in peritonel mcrophges In ortic tissue, TC nd TG mss were significntly incresed in oth HFHC (1.7- nd 2- fold, respectively) nd LFHC groups (1.5- nd 2.2-fold, respectively) compred to controls (Fig. 6A nd 6B). In comprison, nringenin completely prevented the cholesterol-induced deposition of ortic cholesterol nd TG. The HFHC diet significntly incresed expression of the inflmmtory mrkers Tnf, Il1, nd Ccl2 (~2-fold) within the orte (Fig. 6C). Conversely, 15

16 nringenin supplementtion of the HFHC diet normlized inflmmtory cytokine expression. Aorte from the low ft-fed groups were not ville for nlysis of inflmmtory mrkers. Elicited peritonel mcrophges from Ldlr -/- mice fed high-ft, 1.25% cholesterol diet hve een used s n in vivo model of mcrophge fom cell formtion (22). We determined if the nringenin-induced reduction in ortic lipid ccumultion nd inflmmtory gene expression ws relted to decrese in mcrophge fom cell formtion. Peritonel mcrophges from HFHC-fed mice ccumulted significntly more CE (15-fold) nd TG (4-fold), compred to mcrophges from chow-fed mice (Fig. 6D). Incresed lipid mss correlted with enhnced Oil Red O stined lipid droplets (Fig. 6E). In contrst, mcrophges isolted from nringenintreted mice demonstrted >5% reduction in CE nd TG, correlting with mrked reduction in neutrl lipid droplets. Anlysis of inflmmtory cytokine expression in peritonel mcrophges reveled no induction in HFHC-fed mice (Fig. 6F). Nevertheless, nringenin significntly ttenuted the expression of Tnf, Ccl2, nd Ccl3, (-3 to -5%) compred to either chow- or HFHC-fed mice (Fig. 6F). Thus, nringenin s ility to ttenute inflmmtion in peritonel mcrophges my, in prt, e independent of its lipid-lowering properties. Downloded from y guest, on June 19,

17 Discussion Hypercholesterolemi is significnt risk fctor for the development of therosclerosis (26). In Ldlr -/- mice, cholesterol-enrichment of high-ft diets intensifies dyslipidemi nd the inflmmtory response in dipose tissue nd liver, which potentites therogenesis (1,6,7). Moreover, incresing the cholesterol content of low-ft, high sucrose diets, promotes hypercholesterolemi nd therosclerosis in Ldlr -/- mice (16). In the present study, we demonstrte tht ddition of nringenin to cholesterol-rich diets prevents oesity, glucose intolernce nd insulin resistnce. Nringenin mrkedly ttenutes dyslipidemi nd heptic lipid ccumultion. Furthermore, nringenin llevites the cholesterol-induced inflmmtory response in liver, dipose tissue nd ort, collectively resulting in n ttenution of therogenesis. Diets enriched in ft nd/or cholesterol induce heptic stetosis (7,24) primrily consequence of incresed SREBP-1c-induced lipogenesis nd reduced FA oxidtion (5,17). Furthermore, diets rich in simple crohydrtes, such s sucrose or fructose, lso induce heptic TG ccumultion in rodents (9,27). In the present study, the HFHC, LF nd LFHC diets incresed heptic FA synthesis, without compenstory increse in heptic FA oxidtion, Downloded from y guest, on June 19, 218 resulting in mrked heptic stetosis compred to chow-fed mice. Addition of cholesterol to the LF diet resulted in further increse in heptic TG, even in the sence of excess dietry ft. Heptic Sref1c expression ws lso incresed in LFHC-fed mice, reflecting the impct of dietry cholesterol on liver X receptor (LXR)-induced SREBP-1c-stimulted FA synthesis (28). In contrst, nringenin reduced heptic Sref1c expression nd incresed Fgf21, Pgc1 nd Cpt1 mrna, in concert with significntly decresed heptic FA synthesis nd enhnced FA oxidtion. We now demonstrte tht when dded to cholesterol-enriched high-ft diet, nringenin mintins its ility to shift heptic gene expression to prevent heptic TG 17

18 ccumultion. Furthermore, nringenin normlizes hyperinsulinemi nd liver TG in mice fed cholesterol-contining, low-ft, high-sucrose diet (LFHC), primrily through diminished FA synthesis. Heptic pob1 overproduction chrcterizes the dyslipidemi ssocited with insulin resistnce nd incresed heptic lipid drives VLDL production (29,3). The heptic stetosis in HFHC- nd LFHC-fed mice ws ssocited with n over-production of VLDL pob1 nd TG into plsm. Supplementtion of either cholesterol-rich diet with nringenin olished pob1 over-secretion nd ttenuted hyperlipidemi, concomitnt with the striking reduction in heptic TG nd cholesterol. Our results re consistent with the concept tht incresed heptic FA oxidtion s well s decresed hyperinsulinemi-stimulted ftty cid synthesis contriutes to the nringenininduced decrese in heptic stetosis nd VLDL over-production, reduced dipose tissue ccumultion nd normliztion of muscle triglyceride levels. These oservtions re lso consistent with studies in which geneticlly-induced increses in heptic FA oxidtion protects mice from metolic dysregultion. In glol Acc2 -/- mice, in which the synthesis of mlonylcoa is locked thus relieving the inhiition of CPT1, heptic FA oxidtion ws incresed (~15%) Downloded from y guest, on June 19, 218 nd mice were protected from high-ft diet induced heptic stetosis, insulin resistnce nd diposity (31-34). However, this ws not oserved in nother strin of Acc2 -/- mice (35). Furthermore, our results re completely consistent with studies in rts (36) or mice (37) in which heptic-specific over-expression of Cpt1 incresed heptic FA oxidtion nd protected mice from high-ft diet induced heptic stetosis nd the inflmmtory response s well s insulin resistnce nd oesity. In this study, we ssessed energy lnce nd found tht the decrese in ectopic ft deposition in nringenin-treted mice ws ttriuted to ~15% increse in whole-ody energy 18

19 expenditure in mice fed nringenin-supplemented diets. The fct tht EE ws incresed y nringenin, yet physicl ctivity ws similr in nringenin nd non-nringenin-fed mice, suggests tht sustrte oxidtion ws higher with nringenin tretment. Although ft oxidtion nd crohydrte oxidtion re thought to e mutully inhiitory (38), similr RQ mesurements were recorded for nringenin supplemented nd non-supplemented diets, despite higher EE. This suggests tht there is simultneous increse in oth glucose nd ftty cid oxidtion rther thn fuel competition etween ft nd crohydrte in nringenin-treted mice (34). The cholesterol-induced inflmmtory response in oth liver, nd dipose tissue in mice fed the HFHC diet is consistent with previous reports (6,7). The current studies demonstrte tht the inflmmtory response is lso stimulted y ddition of cholesterol to LF diet, model in which tissue TG ccumultion is primrily derived from de novo lipogenesis. However, the mgnitude of inflmmtory response induced y the LFHC diet ws not s gret s in HFHC-fed mice, even though heptic stetosis nd diposity were similr, suggesting tht cholesterol my interct with exogenous FAs to enhnce inflmmtion. Although oth cholesterol-contining diets incresed inflmmtory cytokine expression, we did not distinguish which cell type, Kupffer cell, infiltrting mcrophge or dipocyte, contriuted to inflmmtion in liver nd dipose, Downloded from y guest, on June 19, 218 respectively, s ech cell type cn prticipte in the inflmmtory response (25,39). The ddition of nringenin to either the HFHC or LFHC diets sustntilly reduced mcrophge content of liver nd inflmmtory cytokine expression in oth liver nd dipose tissue. Protection y nringenin my e through its ility to reduce cholesterol- nd FA-induced lipotoxicity. Mechnisms y which dietry cholesterol might promote heptic inflmmtion include incresed FC, s oserved in mice fed either cholesterol-contining diet. Cholesterol feeding hs een shown to increse heptic mitochondril FC resulting in sensitiztion of liver mitochondri to cytokine-medited injury (4). Furthermore, elevted cellulr FC is known to 19

20 stimulte inflmmtory signling pthwys in vitro including mitogen-ctivted protein kinse (MAPK), nd nucler fctor (NF)-κB (41). The ility of nringenin to diminish heptic cholesterol my llevite this inflmmtory stimulus. Incution of dipocytes nd mcrophges with sturted FAs enhnces inflmmtory cytokine secretion, therey linking sturted FAs to the inflmmtory process (42,43). Furthermore, diets rich in sturted ft, including the HFHC diet in the present study, induce mcrophge infiltrtion into dipose tissue nd stimulte the inflmmtory response (44-46). Incresed heptic oxidtion of these FAs in high ft-fed mice over-expressing heptic Cpt1, ttenuted the expression of Tnf, Il6, Il1 nd Ccl3 in liver or dipose tissue (37). Moreover, in mcrophges exposed to excess plmitte, ctivtion of AMPK incresed FA oxidtion nd suppressed inflmmtion (47). Collectively, these results suggest tht nringenin s ntiinflmmtory properties in dipose tissue re secondry to decresed exposure to lipoproteinderived FAs, nd in liver re due to its ility to stimulte FA oxidtion nd/or inhiit FA synthesis. However, direct ffect of nringenin in these tissues on inflmmtory signling pthwys cnnot e ruled out. Nringenin hs een shown to directly inhiit MAPK signling nd NF-κB ctivity in epithelil cells (48). Downloded from y guest, on June 19, 218 Inflmmtion in dipose tissue ssocited with oesity is thought to contriute to insulin resistnce (44,49). In o/o mice, mcrophge infiltrtion into dipose tissue precedes the onset of hyperinsulinemi, suggesting tht insulin resistnce is initited, in prt, y dipose tissue inflmmtion (44). In vitro, TNFα inhiits insulin signling in dipocytes, leding to reduced glucose uptke (5). In the present study, the ility of nringenin to prevent oesity nd cholesterol-induced dipose tissue inflmmtion, my therefore contriute to the improvement in glucose homeostsis nd insulin sensitivity. Two other polyphenolic molecules, resvertrol nd quercetin, when incuted with humn dipocytes, prevented oth TNFα- 2

21 medited inflmmtory cytokine secretion nd inctivtion of the insulin receptor (51). Further mechnistic studies re required to define ny direct contriution of nringenin in dipocytes to its protection from oesity, dipose inflmmtion nd insulin resistnce. Oesity-ssocited chronic low-grde inflmmtion is considered risk fctor for crdiovsculr disese (2,52), nd incresed dietry cholesterol potentites the inflmmtory nd therogenic responses (1,16). In HFHC- nd LFHC-fed mice, incresed liver nd dipose tissue inflmmtion, comined with the development of systemic inflmmtion, contriuted to ccelerted therogenesis, compred to chow- nd LF-fed mice. These mice developed lesions comprised not only of lipid-lden mcrophges, ut lso n undnce of collgen nd SM α- ctin, consistent with the formtion of more complex lesions (53). In nringenin-treted mice the reduced dyslipidemi, improved insulin sensitivity, nd decresed heptic, dipose, nd systemic inflmmtion, collectively contriuted to mrked reduction in therosclerosis. Lesions in nringenin-treted mice were less mture, composed minly of mcrophges, indicting tht in model in which therogenesis is ccelerted y dietry cholesterol, nringenin prevents the progression of lesions to more complex phenotypes. Supplementtion of cholesterol-rich diets with nringenin not only prevented lipid Downloded from y guest, on June 19, 218 ccumultion ut lso inflmmtory cytokine expression within the ort, demonstrting tht the nti-inflmmtory properties of nringenin extend to the rteril wll. Therefore, it ws importnt to estlish if nringenin could inhiit the inflmmtory response t the cellulr level, specificlly in mcrophges, cell tht plys n integrl role in mediting inflmmtion in lesions (54). Elicited peritonel mcrophges from HFHC-fed mice, n in vivo model of fom cell formtion (22), were significntly enriched in cellulr cholesterol nd TG. Prdoxiclly, lipid ccumultion ws not ssocited with incresed expression of inflmmtory mrkers, compred to chow-fed mice indicting tht mcrophge lipid-loding is not necessrily linked to incresed 21

22 inflmmtion. Despite this finding, nringenin significntly ttenuted peritonel mcrophge lipid content nd the expression of Tnf, Ccl2 nd Ccl3. This implies tht nringenin ttenutes inflmmtion in mcrophges not only through its ility to prevent lipid deposition, ut lso vi direct effect on inflmmtory signling. Nringenin is known to modulte inflmmtion in cultured mcrophges through inhiition of NF-κB nd ctivtor protein 1 (AP-1) signling (48,55). However, direct experimentl evidence is required to elucidte the mechnisms, in ddition to lipid-lowering, y which nringenin prevents inflmmtory signling in mcrophges, liver nd dipose tissue. In conclusion, these studies demonstrte tht nringenin prevents metolic dysregultion induced y dietry cholesterol oth in the presence nd sence of dietry ft. Nringenin s potent lipid-lowering properties contriute to reduced hyperlipidemi nd incresed insulin sensitivity. Cholesterol-induced inflmmtion ssocited with oesity nd therosclerosis is prevented y nringenin. These studies lso highlight the possiility tht nringenin hs direct effect on the inflmmtory processes tht potentite therogenesis. Therefore, the eneficil effects of nringenin provide insight into possile therpeutic trgets for preventing therosclerosis ssocited with metolic dysregultion. Downloded from y guest, on June 19,

23 Sources of Funding: This work ws supported y grnts from the Hert nd Stroke Foundtion of Ontrio (HSFO) T-777 nd PG-5967 (to M.W.H.), Cndin Foundtion for Innovtion nd Ontrio Reserch Fund (to R.G.), HSFO Msters Awrd (to J.M.A) nd Cndin Institutes of Helth Reserch-Cnd Grdute Scholrship Doctorl Awrd (to E.E.M.). Disclosures: None Downloded from y guest, on June 19,

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27 VLDL nd LDL polipoprotein B production in miniture pigs. Arterioscler Throm Vsc Biol. 17: Grnton, P. V., C. J. Norley, J. Umoh, E. A. Turley, B. C. Frier, E. G. Nole, nd D. W. Holdsworth. 21. Rpid in vivo whole ody composition of rts using cone em muct. J Appl Physiol. 19: Li, A. C., C. J. Binder, A. Gutierrez, K. K. Brown, C. R. Plotkin, J. W. Pttison, A. F. Vlledor, R. A. Dvis, T. M. Willson, J. L. Witztum, W. Plinski, nd C. K. Glss. 24. Differentil inhiition of mcrophge fom-cell formtion nd therosclerosis in mice y PPARlph, et/delt, nd gmm. The Journl of clinicl investigtion. 114: Wilcox, L. J., P. H. Brrett, R. S. Newton, nd M. W. Huff ApoB1 secretion from HepG2 cells is decresed y the ACAT inhiitor CI-111: n effect ssocited with enhnced intrcellulr degrdtion of ApoB. Arterioscler Throm Vsc Biol. 19: Wouters, K., P. J. vn Gorp, V. Bieghs, M. J. Gijels, H. Duimel, D. Lutjohnn, A. Kerksiek, R. vn Kruchten, N. Med, B. Stels, M. vn Bilsen, R. Shiri-Sverdlov, nd M. H. Hofker. 28. Dietry cholesterol, rther thn liver stetosis, leds to heptic Downloded from y guest, on June 19, 218 inflmmtion in hyperlipidemic mouse models of nonlcoholic stetoheptitis. Heptology. 48: Surmi, B. K., nd A. H. Hsty. 28. Mcrophge infiltrtion into dipose tissue: initition, propgtion nd remodeling. Future lipidology. 3: Snidermn, A. D., T. Pedersen, nd J. Kjekshus Putting low-density lipoproteins t center stge in therogenesis. Am J Crdiol. 79: Roncl-Jimenez, C. A., M. A. Lnsp, C. J. Rivrd, T. Nkgw, L. G. Snchez- Lozd, D. Jll, A. Andres-Hernndo, K. Tne, M. Mdero, N. Li, C. Cicerchi, K. Mc 27

28 Fnn, Y. Y. Sutin, nd R. J. Johnson Sucrose induces ftty liver nd pncretic inflmmtion in mle reeder rts independent of excess energy intke. Metolism. 6: Rep, J. J., G. Ling, J. Ou, Y. Bshmkov, J. M. Loccro, I. Shimomur, B. Shn, M. S. Brown, J. L. Goldstein, nd D. J. Mngelsdorf. 2. Regultion of mouse sterol regultory element-inding protein-1c gene (SREBP-1c) y oxysterol receptors, LXRlph nd LXRet. Genes Dev. 14: Adiels, M., S. O. Olofsson, M. R. Tskinen, nd J. Boren. 28. Overproduction of very low-density lipoproteins is the hllmrk of the dyslipidemi in the metolic syndrome. Arterioscler Throm Vsc Biol. 28: Adiels, M., M. R. Tskinen, C. Pckrd, M. J. Cslke, A. Soro-Pvonen, J. Westerck, S. Vehkvr, A. Hkkinen, S. O. Olofsson, H. Yki-Jrvinen, nd J. Boren. 26. Overproduction of lrge VLDL prticles is driven y incresed liver ft content in mn. Dietologi. 49: Au-Elheig, L., M. M. Mtzuk, K. A. Ao-Hshem, nd S. J. Wkil. 21. Continuous ftty cid oxidtion nd reduced ft storge in mice lcking cetyl-coa croxylse 2. Downloded from y guest, on June 19, 218 Science. 291: Au-Elheig, L., W. Oh, P. Kordri, nd S. J. Wkil. 23. Acetyl-CoA croxylse 2 mutnt mice re protected ginst oesity nd dietes induced y high-ft/highcrohydrte diets. Proc Ntl Acd Sci U S A. 1: Au-Elheig, L., H. Wu, Z. Gu, R. Bressler, nd S. J. Wkil Acetyl-CoA croxylse 2-/- mutnt mice re protected ginst ftty liver under high-ft, highcrohydrte dietry nd de novo lipogenic conditions. J Biol Chem. 287:

29 34. Choi, C. S., D. B. Svge, L. Au-Elheig, Z. X. Liu, S. Kim, A. Kulkrni, A. Distefno, Y. J. Hwng, R. M. Reznick, R. Codell, D. Zhng, G. W. Cline, S. J. Wkil, nd G. I. Shulmn. 27. Continuous ft oxidtion in cetyl-coa croxylse 2 knockout mice increses totl energy expenditure, reduces ft mss, nd improves insulin sensitivity. Proc Ntl Acd Sci U S A. 14: Hoehn, K. L., N. Turner, M. M. Swrrick, D. Wilks, E. Preston, Y. Phu, H. Joshi, S. M. Furler, M. Lrnce, B. D. Hegrty, S. J. Leslie, R. Pickford, A. J. Hoy, E. W. Kregen, D. E. Jmes, nd G. J. Cooney. 21. Acute or chronic upregultion of mitochondril ftty cid oxidtion hs no net effect on whole-ody energy expenditure or diposity. Cell Met. 11: Stefnovic-Rcic, M., G. Perdomo, B. S. Mntell, I. J. Sipul, N. F. Brown, nd R. M. O'Doherty. 28. A moderte increse in crnitine plmitoyltrnsferse 1 ctivity is sufficient to sustntilly reduce heptic triglyceride levels. Am J Physiol Endocrinol Met. 294: E Orelln-Gvld, J. M., L. Herrero, M. I. Mlndrino, A. Pned, M. Sol Rodriguez- Pen, H. Petry, G. Asins, S. Vn Deventer, F. G. Hegrdt, nd D. Serr Moleculr Downloded from y guest, on June 19, 218 therpy for oesity nd dietes sed on long-term increse in heptic ftty-cid oxidtion. Heptology. 53: Rndle, P. J., P. B. Grlnd, E. A. Newsholme, nd C. N. Hles The glucose ftty cid cycle in oesity nd mturity onset dietes mellitus. Ann N Y Acd Sci. 131: Hung, W., A. Metlkunt, N. Dedousis, P. Zhng, I. Sipul, J. J. Due, D. K. Scott, nd R. M. O'Doherty. 21. Depletion of liver Kupffer cells prevents the development of dietinduced heptic stetosis nd insulin resistnce. Dietes. 59:

30 4. Mri, M., F. Cllero, A. Colell, A. Morles, J. Clleri, A. Fernndez, C. Enrich, J. C. Fernndez-Chec, nd C. Grci-Ruiz. 26. Mitochondril free cholesterol loding sensitizes to TNF- nd Fs-medited stetoheptitis. Cell Met. 4: Li, Y., R. F. Schwe, T. DeVries-Seimon, P. M. Yo, M. C. Gerod-Ginnone, A. R. Tll, R. J. Dvis, R. Flvell, D. A. Brenner, nd I. Ts. 25. Free cholesterol-loded mcrophges re n undnt source of tumor necrosis fctor-lph nd interleukin-6: model of NF-kppB- nd mp kinse-dependent inflmmtion in dvnced therosclerosis. J Biol Chem. 28: Sugnmi, T., K. Tnimoto-Koym, J. Nishid, M. Itoh, X. Yun, S. Mizuri, H. Kotni, S. Ymok, K. Miyke, S. Aoe, Y. Kmei, nd Y. Ogw. 27. Role of the Toll-like receptor 4/NF-kppB pthwy in sturted ftty cid-induced inflmmtory chnges in the interction etween dipocytes nd mcrophges. Arterioscler Throm Vsc Biol. 27: Anderson, E. K., A. A. Hill, nd A. H. Hsty Steric cid ccumultion in mcrophges induces toll-like receptor 4/2-independent inflmmtion leding to endoplsmic reticulum stress-medited poptosis. Arterioscler Throm Vsc Biol. 32: Downloded from y guest, on June 19, Xu, H., G. T. Brnes, Q. Yng, G. Tn, D. Yng, C. J. Chou, J. Sole, A. Nichols, J. S. Ross, L. A. Trtgli, nd H. Chen. 23. Chronic inflmmtion in ft plys crucil role in the development of oesity-relted insulin resistnce. The Journl of clinicl investigtion. 112: Weiserg, S. P., D. McCnn, M. Desi, M. Rosenum, R. L. Leiel, nd A. W. Ferrnte, Jr. 23. Oesity is ssocited with mcrophge ccumultion in dipose tissue. The Journl of clinicl investigtion. 112:

31 46. Coenen, K. R., M. L. Gruen, A. Chit, nd A. H. Hsty. 27. Diet-induced increses in diposity, ut not plsm lipids, promote mcrophge infiltrtion into white dipose tissue. Dietes. 56: Glic, S., M. D. Fullerton, J. D. Schertzer, S. Sikkem, K. Mrcinko, C. R. Wlkley, D. Izon, J. Honeymn, Z. P. Chen, B. J. vn Denderen, B. E. Kemp, nd G. R. Steinerg Hemtopoietic AMPK et1 reduces mouse dipose tissue mcrophge inflmmtion nd insulin resistnce in oesity. The Journl of clinicl investigtion. 121: Yng, J., Q. Li, X. D. Zhou, V. P. Kolosov, nd J. M. Perelmn Nringenin ttenutes mucous hypersecretion y modulting rective oxygen species production nd inhiiting NF-kppB ctivity vi EGFR-PI3K-Akt/ERK MAPKinse signling in humn irwy epithelil cells. Mol Cell Biochem. 351: Hotmisligil, G. S., P. Perldi, A. Budvri, R. Ellis, M. F. White, nd B. M. Spiegelmn IRS-1-medited inhiition of insulin receptor tyrosine kinse ctivity in TNF-lphnd oesity-induced insulin resistnce. Science. 271: Hotmisligil, G. S., D. L. Murry, L. N. Choy, nd B. M. Spiegelmn Tumor Downloded from y guest, on June 19, 218 necrosis fctor lph inhiits signling from the insulin receptor. Proc Ntl Acd Sci U S A. 91: Chung, C. C., K. Mrtinez, G. Xie, A. Kennedy, A. Bumrungpert, A. Overmn, W. Ji, nd M. K. McIntosh. 21. Quercetin is eqully or more effective thn resvertrol in ttenuting tumor necrosis fctor-{lph}-medited inflmmtion nd insulin resistnce in primry humn dipocytes. The Americn journl of clinicl nutrition. 92: Berg, A. H., nd P. E. Scherer. 25. Adipose tissue, inflmmtion, nd crdiovsculr disese. Circ Res. 96:

32 53. Whitmn, S. C. 24. A prcticl pproch to using mice in therosclerosis reserch. Clin Biochem Rev. 25: Ts, I. 21. Mcrophge deth nd defective inflmmtion resolution in therosclerosis. Nt Rev Immunol. 1: Bodet, C., V. D. L, F. Epifno, nd D. Grenier. 28. Nringenin hs nti-inflmmtory properties in mcrophge nd ex vivo humn whole-lood models. J Periodontl Res. 43: Downloded from y guest, on June 19,

33 Figure Legends Fig. 1. Nringenin meliortes dyslipidemi in mice fed cholesterol-rich diets. Mle Ldlr -/- mice were fed either chow, high-ft/high-cholesterol (HFHC) ± Nringenin (Nr), low-ft (LF), or low-ft/high-cholesterol (LFHC) ± Nr for 12 weeks. (A) nd (B) Fst Performnce Liquid Chromtogrphy (FPLC) of plsm. Cholesterol nd TG concentrtions were mesured in eluted frctions (n= 3 mice/group). (C) TG secretion t nd 12 min post i.p. injection of tyloxpol (n= 5-6/group/time point). Inset, TG secretion rte expressed s mmol/l/h. (D) Secretion of VLDL/IDL pob1 t 6 nd 12 min post i.p injection of tyloxpol nd [ 35 S]methionine (n= 5-6/group/time point). A VLDL/IDL frction ws isolted y ultrcentrifugtion. Inset, VLDL/IDL pob1 secretion rte expressed s ritrry phosphorimger volume units (AU) per h. Vlues re the men ± SEM. Different letters re sttisticlly different (P<.5). Fig. 2. Heptic cholesterol nd FA metolism in cholesterol-fed nimls with nringenin tretment. (A) Heptic totl cholesterol (whole r), cholesteryl ester (lck r), nd free cholesterol (open r). Vlues re the men ± SEM. Different letters re sttisticlly different (P<.5) for ech lipid prmeter. (B) Heptic triglyceride mss. (C) nd (D) Representtive Downloded from y guest, on June 19, 218 fluorescence microgrphs of liver sections stined with Oil Red O nd Hoechst to visulize neutrl lipid droplets (red) nd nuclei (lue). Scle r = 75 μm. (E) nd (F) Heptic mrna expression (n= 1-12/group). (G) Cholesterol synthesis in the liver (n= 6-8/group) determined y incorportion of [1-14 C]cetic cid into cholesterol. (H) FA synthesis in the liver (n= 6-8/group) otined 6 min post injection i.p. with [1-14 C]cetic cid. (I) Heptic FA oxidtion (n= 6-8/group) determined y [ 3 H]plmitte conversion to H 2 O. Vlues re the men ± SEM. (J) Energy expenditure determined y indirect clorimetry (CLAMS system) during the light nd 33

34 drk cycles (7: pm 7: m). Mesurements were collected every 1 minutes nd ech r represents the men ± SEM, normlized to len ody mss (LBM) for the entire drk cycle or totl 24 h period (n=6/group). Different letters re sttisticlly different (P<.5). Asterisk (*) indictes sttisticl significnce etween Chow nd LF (P<.5). Fig. 3. Prevention of heptic inflmmtion in cholesterol-fed mice supplemented with nringenin. (A) nd (B) Heptic mrna expression (n= 1-12/group). (C) nd (D) Representtive photomicrogrphs of liver, stined with MAC-2 ntiody for the presence of mcrophges (drk rown stin, indicted y rrows). Scle r = 1 μm. Vlues re the men ± SEM. Different letters re sttisticlly different (P<.5). Fig. 4. Reduced dipose tissue expnsion nd inflmmtion, improved glucose homeostsis nd enhnced insulin sensitivity in nringenin-treted mice. (A) Adiposity index expressed s epididyml ft pd weight (g) normlized to ody weight (g). (B) nd (C) Adipocyte dimeter distriution in epididyml ft expressed s the percentge of totl dipocytes counted versus the dipocyte dimeter (μm). (D) nd (E) Adipose tissue mrna expression (n= 8/group). (F) Glucose tolernce tests (GTT) were performed fter 6 h fst y i.p. injection of 1 g/kg glucose (n= 1-12/group). Inset grph, solute re under the curve Downloded from y guest, on June 19, 218 (AUC), expressed s glucose (mmol/l) x 12 minutes. (G) Insulin tolernce tests (ITT) were performed fter 5 h fst y i.p. injection of insulin (.6 IU/kg) (n= 1-12/group). Inset grph, re under the curve (AUC), expressed s glucose (mmol/l) x 6 minutes. Vlues re the men ± SEM. Different letters re sttisticlly different (P<.5). Asterisk (*) indictes sttisticl significnce etween Chow nd LF (P<.5). Fig. 5. Prevention of therosclerosis in cholesterol-fed mice in the ort y nringenin supplementtion. (A) nd (B) Plque re (μm 2 x 1 5 ) in seril sections of ortic sinus stined 34

35 with Oil Red O (n= 8-1/group). Representtive photomicrogrphs of ortic sinus stined with Oil Red O nd counterstined with hemtoxylin. (C) nd (D) Atherosclerosis development in the ortic rch, (% Sudn IV-stined plque re per totl surfce re in scending nd descending ort) (n= 6-8/group). (A-D) Vlues from individul mice re represented y symols nd the men lesion re is depicted s single horizontl line. (E) nd (F) Grphicl representtion of MOMA-2 (mcrophges), collgen (from trichrome), nd SM α-ctin-positively stined re expressed s % of stined lesion re. Vlues re the men ± SEM. Different letters re sttisticlly different (P<.5). Fig. 6. Prevention of ortic lipid ccumultion nd inflmmtion in cholesterol-fed mice treted with nringenin. (A) nd (B) Totl cholesterol nd triglyceride concentrtions in whole orte (n= 6-8/group). (C) mrna expression in whole ort (n= 6-7/group). (D) Mle Ldlr -/- mice were fed chow or HFHC ± Nr for 12 weeks (n= 16/group), followed y i.p. injection with thioglycollte. After 5 dys, peritonel mcrophges were hrvested nd nlyzed for cellulr lipid concentrtions. (E) Representtive photomicrogrphs of peritonel mcrophges, stined with Oil Red O nd hemtoxylin. Scle r = 1 μm. (F) Peritonel mcrophge mrna expression (n= 1-14/group). Vlues re the men ± SEM. Different letters re sttisticlly Downloded from y guest, on June 19, 218 different (P<.5). 35

36 Metolic Vrile Finl Body Weight (g) Cloric Intke (kcl/dy/ mouse) Plsm Cholesterol (mmol/l) Plsm TG (mmol/l) Plsm Insulin (ng/ml) Blood Glucose (mmol/l) Plsm Leptin (ng/ml) Plsm SAA (μg/ml) Bile Acids (gll ldder) (µmol/ml) Cholesterol Asorption (%) Triglyceride Asorption (%) Activity (counts/hour) Tle 1. Prmeters in Mle Ldlr -/- Mice After 12 Weeks On Diet Chow HFHC HFHC + Nr LF LFHC LFHC + Nr ± ± ± ±.59 * ± ± ± ± ± ±.48* ± ± ± ± ± 1.95 c ±.98 * 46.8 ± ± 2.5 c.66 ± ± ±.24 c 1.96 ±.15 * 1.83 ± ±.7.2 ±.4.76 ± ±.3.47 ±.4 *.48 ± ± ± ± ±.4 8. ±.45 * 8.39 ± ± ± ± ± ± 1.19 * 7.55 ± ± ± ± ± ± 1.72 * 23.4 ± ± ± 6 35 ± ± ± ± ± 7 48 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 194 ND 3148 ± ± 93 Downloded from y guest, on June 19, 218 RQ.92 ±.1.82 ±.1.85 ±.2 ND.96 ±.4.95 ±.1 Dt re given s men ± SEM (n=1-12/group) Different letters indicte sttisticl significnce within either study ((Chow, HFHC, nd HFHC + Nr) or (LF, LFHC, nd LFHC + Nr)) (P<.5) Asterisk (*) indictes sttisticl significnce etween Chow nd LF (P<.5) HFHC; high-ft, high-cholesterol, Nr; nringenin, LF; low-ft, LFHC; low-ft, high-cholesterol, SAA; serum myloid A, TG; triglyceride; Activity; multory plus stereotypic movement em reks over 24 hours, expressed s counts per hour. RQ; respirtory quotient, ND; not determined 36

37 A B C D Figure 1 Cholesterol (μg/ml) Triglyceride (μg/ml) Triglyceride (mmol/l) ApoB1 (Phosphorimger Volume AU x 1 3 ) FPLC Cholesterol VLDL LDL HDL Frction Numer VLDL FPLC Triglyceride Frction Numer 6 Secretion Rte Triglyceride Secretion Time (min) ApoB1 Secretion 25 Secretion Rte Time (min) Chow HFHC HFHC + Nr Cholesterol (μg/ml) Triglyceride (μg/ml) Triglyceride (mmol/l) ApoB1 (Phosphorimger Volume AU x 1 3 ) 16 FPLC Cholesterol 14 VLDL 12 1 LDL HDL Frction Numer VLDL FPLC Triglyceride Frction Numer 6 Secretion Rte Triglyceride Secretion Time (min) ApoB1 Secretion Secretion Rte 6 12 Time (min) LF LFHC LFHC + Nr Downloded from y guest, on June 19,

38 Figure 2 Lipid Mss (μg/mg Tissue) A Heptic Cholesterol Cholesteryl Ester Free Cholesterol c * B Lipid Mss (μg/mg Tissue) Heptic Triglyceride c * C D E G pmoles of cetic cid into cholesterol/g tissue I mrna of interest/gpdh (fold chnge of control) nmol plmitte oxidized/ min/mg tissue protein Chow HFHC HFHC + Nr Heptic Gene Expression Heptic Cholesterol Synthesis Heptic Ftty Acid Oxidtion c c * Chow HFHC HFHC + Nr F mrna of interest/gpdh (fold chnge of control) H μmoles of cetic cid into ftty cid/mg tissue J 12 1 Energy Expenditure (kcl/kg LBM/h) LF LFHC LFHC + Nr Heptic Gene Expression Heptic Ftty Acid Synthesis * Energy Expenditure Drk Totl Drk Totl LF LFHC LFHC + Nr Downloded from y guest, on June 19,

39 Figure 3 A B mrna of interest/gpdh (fold chnge of control) mrna of interest/gpdh (fold chnge of control) Tnf Il1 Il6 Chow HFHC HFHC + Nr Heptic Gene Expression Ccl2 c Ccl3 Heptic Gene Expression LF LFHC LFHC + Nr F4/8 S1/2 Tnf Il1 Il6 Ccl2 Ccl3 F4/8 S1/2 C D MAC-2 Chow HFHC HFHC + Nr MAC-2 LF LFHC LFHC + Nr Downloded from y guest, on June 19,

40 Figure 4 A B C Epididyml Ft (g)/ Body Weight (g) Adiposity Index * % of Totl Adipocytes Counted Adipocyte Size % of Totl Adipocytes Counted Adipocyte Size mrna of interest/gpdh (fold chnge of control) D F Blood Glucose (mmol/l) G Blood Glucose (mmol/l) 3 GTT Tnf Il1 Il6 Ccl2 Ccl3 F4/ Adipose Tissue Gene Expression Time (min) ITT Time (min) 25 AUC AUC mrna of interest/gpdh (fold chnge of control) Chow HFHC HFHC + Nr Dimeter (μm) E Blood Glucose (mmol/l) Blood Glucose (mmol/l) GTT Dimeter (μm) Tnf Il1 Il6 Ccl2 Ccl3 F4/ Adipose Tissue Gene Expression Time (min) ITT Time (min) AUC AUC LF LFHC LFHC + Nr Downloded from y guest, on June 19, 218 4

41 Figure 5 A 8 Aortic Sinus Quntittion of Atherosclerosis B 8 Aortic Sinus Quntittion of Atherosclerosis Plque Are (μm 2 x 1 5 ) c Plque Are (μm 2 x 1 5 ) c -2 Chow HFHC HFHC + Nr -2 LF LFHC LFHC + Nr C Atherosclerosis (% of totl surfce re) E % of Stined Lesion Are En fce Quntittion of Atherosclerosis Chow HFHC HFHC + Nr Plque Composition MOMA-2 Collgen SM α-ctin Chow HFHC HFHC + Nr c % of Stined Lesion Are D Atherosclerosis (% of totl surfce re) F En fce Quntittion of Atherosclerosis LF LFHC LFHC + Nr Plque Composition MOMA-2 Collgen LF LFHC LFHC + Nr SMC α-ctin SM α-ctin c Downloded from y guest, on June 19,

42 Figure 6 Cholesterol (μg/mg Tissue) mrna of interest/gpdh (fold chnge of control) A C E Aortic Lipids Totl Cholesterol Triglyceride TG (μg/mg Tissue) Cholesterol (μg/mg Tissue) Chow HFHC HFHC + Nr LF LFHC LFHC + Nr Tnf Aortic Gene Expression Il1 Ccl2 μg/mg cell protein B D Totl Cholesterol Aortic Lipids Triglyceride Lipid Mss in Peritonel Mcrophges c Totl Cholesterol c Cholesteryl Ester c Triglyceride TG (μg/mg Tissue) Downloded from y guest, on June 19, 218 F mrna of interest/gpdh (fold chnge of control) Chow HFHC HFHC + Nr Gene Expression in Peritonel Mcrophges Tnf Il1 Il6 Ccl2 Ccl3 Chow HFHC HFHC + Nr 42