Mesozeaxanthin protects the liver and reduces cardio-metabolic risk factors in an insulin resistant rodent model

Transcription

1 FOOD & NUTRITION RESEARCH, 217 VOL. 61, ARTICLE Mesozexnthin protets the liver nd redues rdio-metoli risk ftors in n insulin resistnt rodent model Kzim Shin, Ceml Orhn, Ftih Akdemir, Mehmet Tuzu, Nurhn Shin, Ismet Yilmz d, Shkir Ali e, Jynt Deshpnde f nd Vijy Juturu f Deprtment of Animl Nutrition, Fulty of Veterinry Siene, Firt University, Elzig, Turkey; Deprtment of Nutrition, Fulty of Fisheries, Inonu University, Mlty, Turkey; Division of Biology, Fulty of Siene, Firt University, Elzig, Turkey; d Deprtment of Phrmology, Fulty of Phrmy, Inonu University, Mlty, Turkey; e Deprtment of Biohemistry, Fulty of Siene, Jmi Hmdrd, New Delhi, Indi; f Reserh nd Development, OmniAtive Helth Tehnologies In., Morristown, NJ, USA ABSTRACT Bkground: Mesozexnthin (MZ) is mulr rotenoid whih hs een reported to hve numer of phrmologil properties, inluding the ntioxidnt, nd ntirinogeni property, nd hs een stted to derese the heptoyte lipid ontent. Ojetive: In this study, we investigted the effet of MZ on rdio-metoli helth risk (CMHR) nd its prole mehnisms of tion in rts fed high-ft diet (HFD). Design: Rts were rndomly divided into four groups onsisting of (i) Control, (ii) MZ, (iii) HFD, nd (iv) HFD+MZ. Results: MZ tretment inresed the ntioxidnt enzyme tivities nd helped improve the liver funtion. The tretment llevited CMHR nd deresed the level of nuler ftor kpp B (NF-κB p65) nd tumor nerosis ftor-lph (TNF-α). The levels of hepti peroxisome prolifertortivted reeptor gmm (PPAR-γ), phosphorylted insulin reeptor sustrte 1 (p-irs-1), β,βrotene 9,1 -oxygense 2 (BCO2) nd nuler ftor erythroid 2-relted ftor 2 (Nrf2), whih derese in HFD rts, were found to e signifintly higher in MZ supplemented nimls. Conlusion: MZ hs ntioxidnt nd nti-inflmmtory properties nd n is reported in this study toprotet ginst ftty liver nd rdio-metoli syndrome, possily through regultion of PPAR-γ, IRS-1, Nrf2 nd NF-κB proteins, in n insulin-resistnt rodent model. ARTICLE HISTORY Reeived 24 My 217 Aepted 24 June 217 KEYWORDS High-ft diet; insulin resistne; liver; mesozexnthin; rts The rdio-metoli syndrome (CMS) hs een reported to e relted to the risk of developing type 2 dietes mellitus (T2DM), rdiovsulr disese (CVD) nd stroke [1 4]. The syndrome, hrterized ythelusteringoftlestthreeoffiveofthefollowing medil onditions (i) entrl or norml oesity; (ii) high lood pressure (BP); (iii) rised fsting plsm gluose; (iv) high plsm triglyerides (TG); nd (v) low level of high-density lipoprotein holesterol (HDL-C) hs een reported to e used y exess diposity s usul key ftor [3,4]. Ftty liver in oese individuls is one of the most ritil rdiometoli helth risk (CMHR) in insulin resistne, thus leding to CMS [5,6]. In the liver, existene of someftisnorml,utninreseinftontentto more thn 5 1% of the liver weight hs een ssoited with the ftty liver, whih n e loholi or non-loholi nd my led to serious omplitions relted to lipid imlne nd inresed free ftty ids in the lood [7,8]. Mesozexnthin [MZ; (3R,3S)-dihydroxy-, -rotene-3,3-diol] is non-provitmin A rotenoid in green nd yellow vegetles whih hs een found to e effetive in ging mul to mintin its struturl density [9,1]. The pigment resides diretly over the entre of mul where there is the strongest need for protetion ginst tini lue light. The pigment is synthesized in the retin from ingested lutein. If tken s supplement, MZ is reported to e sored into the lood strem nd effetively inrese the mulr pigment onentrtion. The mulr pigment hs een reported to hve vrious protetive effets whih inlude ntioxidnt, hemo-protetive, nti-mutgeni nd nti-rinogeni effets, owing to its singlet oxygen quenhing ility nd its inhiitory effet on speifi CYP4 proteins, esides its potentil to indue phse II enzymes [9 12]. In vitro, MZ hs een demonstrted to redue heptoyte lipid ontent [1,13,14], whih led us to speulte on the potentil of MZ in reduing ftty liver, CMHR. We tested this hypothesis in rt model of insulin resistne (IR) indued CONTACT Kzim Shin nshinkm@yhoo.om Veterinry Fulty, Firt University, Elzig, Turkey 217 The Author(s). Pulished y Inform UK Limited, trding s Tylor & Frnis Group. This is n Open Aess rtile distriuted under the terms of the Cretive Commons Attriution Liense ( whih permits unrestrited use, distriution, nd reprodution in ny medium, provided the originl work is properly ited.

2 2 K. SAHIN ET AL. y high-ft diet (HFD) in rodents. This study showed signifint orreltion etween MZ supplementtion nd deresed the risk of ftty liver nd CMS nd hs implition in the mngement of ftty liver nd insulin resistne y regulting PPAR-γ,IRS-1,NF-κB nd Nrf2 pthwys. Mteril nd methods Animls nd diets Eight weeks old mle Sprgue-Dwley rts (18 ± 2 g) were used in this study. The rts were kept t 22 ± 2 C, 55 ± 5% humidity nd 12/12 h light/drk yle nd provided rt how (Reserh Diet, NJ, USA) nd wter d liitum. All rts reeived humne re ording to the stndrds defined in the Guide for the Cre nd Use of Lortory Animls prepred y the Ntionl Ademy of Sienes nd pulished y Ntionl Institute of Helth. The study ws permitted y the Ethis Committee of the University of Inonu, Mlty, Turkey. The omposition of ontrol nd high-ft diet [15] is shown in detil in Tle 1. For indution of oesity, nimls were fed with HFD for 12 weeks nd ompred with norml diet fed rts. Experimentl proedures Twenty-eight nimls were limtized for two weeks nd rndomly divided into four groups (n = 7), onsisting of (i) ontrol group (untreted rts, fed with norml how, 12% of lories s ft, for 12 weeks), (ii) MZ supplemented group (fed norml how nd treted with MZ, mg/kg BW, for 12 weeks), (iii) HFD group (rts fed high-ft diet, 42% of lories s ft, for 12 weeks), nd (iv) HFD+MZ group (treted with high-ft diet nd MZ for 12 weeks). MZ (5%) ws dissolved nd delivered in sunflower oil orlly y gvge. The seletion of MZ dose ( mg/kg BW) ws sed on previously pulished Tle 1. Composition of diets (g/kg diet) fed to rts. Control HFD Csein Strh Surose Soyen oil 7. Beef tllow 4. Cellulose.. Vitmin minerl premix* L-ysteine Choline itrtrte Notes: *The vitmin minerl premix provides the following (per kg): ll-trnsretinyl ette, 1.8 mg; holeliferol,.25 mg; ll-r--toopherol ette, 12,5 mg; mendione (mendione sodium isulfte), 1.1 mg; rioflvin, 4.4 mg; thimine (thimine mononitrte), 1.1 mg; vitmin B-6, 2.2 mg; niin, 35 mg; C-pntothente, 1 mg; vitmin B-12,.2 mg; foli id,.55 mg; d-iotin,.1 mg. mngnese (from mngnese oxide), 4 mg; iron (from iron sulfte), 12.5 mg; zin (from zin oxide), 25 mg; opper (from opper sulfte), 3.5 mg; iodine (from potssium iodide),.3 mg; selenium (from sodium selenite),.15 mg; holine hloride, 175 mg. studies, where the ompound hs een shown to exhiit signifint ntioxidnt effet in rodents [11,16]. The ontrol rts in this study reeived similr mounts of sunflower oil y gvge. MZ ws supplied y Omni Ative Helth Tehnologies Pvt. Ltd. (Mumi, Indi). The omposition onsisted of 92% (trns,3r,3 S,meso)-zexnthin out of the totl xnthophyll ontent. The mount of (trns, R,R)-zexnthin ws found to e 4% nd the mount of (trns, R,R)-lutein ws found to e 4% sed on pek res. The omposition of other ingredients ws enntiomers, metolites, esters, slts, derivtives either lone, or in omintions thereof, long with one or more food grde exipients. The result of hromtogrphi nlysis of MZ is shown in Figure 1. At the end of the study, the lood ws olleted fter n overnight fst nd ll nimls were srified y ervil dislotion. The viserl ft nd liver smples were removed nd weighed fter srifiing the nimls. Biohemil estimtions Serum ws prepred y entrifuging the lood t 3 g for 1 min nd used for the nlyses of gluose, insulin, nd mlondildehyde (MDA). Serum prmeters were mesured using n utomti nlyser (Smsung LABGEO PT1, Smsung Eletronis Co, Suwon, Kore). Repetility nd devie/method extness of LABGEO PT1 ws heked ording to the IVR-PT6 guideline. Serum insulin, leptin, nd diponetin were mesured with the Rt Insulin, Leptin nd Adiponetin Kits (Lino Reserh In., St Chrles, MO, USA) y ELISA (Elx-8, Bio-Tek Instruments In., Vermont, USA) respetively. The sensitivity of the ssys ws.22,.36 nd.15 ng/ml for insulin, leptin, nd diponetin respetively. The interssy nd intr-ssy oeffiients of vrition were 4.1 nd 6.7% for insulin, 2.9 nd 5.8% for leptin, nd 2.5 nd 6.4% for diponetin respetively. Insulin resistne ws ssessed using the homeostsis model ssessment for insulin resistne (HOMA-IR), whih ws lulted using the following formul: gluose (mg/dl) insulin (μiu/ml)/22.5. The mulr rotenoids in the hepti tissue (MZ, Z, nd L) were determined y high-performne liquid hromtogrphy (HPLC) (Shimdzu, Tokyo, Jpn) using Shimdzu UV-vis (UV-visile Spetrophotometer) SPD-1AVP detetor nd C18 ODS-3, 5 µm, mm olumn. Briefly, 3 mg of the liver tissue ws dded to mixture of µl 12% pyrogllol in ethnol, 2 µl ehinenone (43 µg/dl) s internl stndrd, 2 µl 3% KOH, 6 µl utylted hydroxytoluene (BHT) (1 mg/ml) nd 1 ml ethnol. The smples were then sponified t 37 C for 2 h. The mixture ws extrted twie with 3 ml ether: hexne (2:1,

3 FOOD & NUTRITION RESEARCH 3 Figure 1. Chromtogrphy (HPLC) of mesozexnthin. y vol.). The extrt ws evported to dryness under nitrogen. The residue ws dissolved in 1 µl ethnol, nd µl smple ws used for HPLC nlysis [17]. All extrts were proteted on the ie nd from light. In the HPLC nlysis, the olumn ws pssed through with methnol:etonitrile:dihloromethne:wter (7:7:2:.16) ontining 2 mmol/l mmonium ette t flow rte of 1 ml per min nd detetion t 4 nm. The hepti MDA ontent ws determined y HPLC ording to the method desried y Krtepe [18] using Shimdzu UV vis SPD-1 AVP detetor, CTO-1 AS VP olumn nd moile phse onsisting of 3 mm KH 2 PO 4 nd methnol (82.5: 17.5, v/v, ph 3.6) t flow rte of 1.2 ml/min. Column effluents were monitored t 2 nm. The volume of the smple injeted into HPLC ws 2 μl. Liver homogente (1%, w/v) ws prepred in 1 mm phosphte uffer (ph 7.4), entrifuged t 13, g for 1 min t 4 C. The resulting superntnt ws olleted nd stored t 8 C for the estimtion of MDA. Totl ntioxidnt pity (TAC) ws determined y following the redution of drk lue-green 2,2 -zino-is 3- ethylenzothizoline-6-sulfonte (ABTS) dye into olourless form y ntioxidnts [19]. The dye ABTS ws oxidized y inuting with potssium persulfte. Briefly, 1 mg ATBS ws dissolved in 1 ml of n queous solution of 2.5 mmol/l potssium persulfte nd llowed to stnd in the drk t room temperture for one to four hours efore use. For use, ABTS oxidized stok solution ws diluted with deionized wter to n sorne of.7 t 734 nm. After the ddition of 1 ml of diluted ABTS oxidized to 1 µl of serum, the sorne reding ws tken 1 minutes fter initil mixing. The ssy hs een reported for its exellent preision (within- nd etween-lortory preision vlues re reported to e lower thn 3%). The results were expressed in mmol Trolox equivlents/l. Totl superoxide dismutse (SOD) tivity (ytosoli nd mitohondril) in the homogenized tissue (in 2 mm HEPES (N-2 hydroxy-ethyl piperzine-n -2-ethnesulfoni id) uffer, 1 mm ethylene glyol tetreti id, 21 mm mnnitol, 7 mm surose, ph 7.2 per g of tissue) ws mesured using ommeril kit (Cymn Chemil, Ann Aror, MI, USA) ording to the mnufturer s instrutions. The superntnt ws olleted fter entrifugtion t 12. g for 2 min t 4 C. The superntnt ws deslted y pssge through Sephdex G-25 olumn. The SOD smples were lso treted with mixture of ethnol-hloroform (2:1, v/v) nd distilled wter to eliminte hemogloin nd red lood ells nd then the sorption ws red on plte reder (Biotek Instruments, In., Vermont, USA) t 4 nm. Results were expressed s units per mg of protein (U/mg of protein). Ctlse (CAT) tivity ws lso determined in the homogenized tissue (in old uffer ontining mm potssium phosphte, 1 mm EDTA, ph 7, per g of tissue) using ommeril kit (Cymn Chemil, Ann Aror, MI, USA) ording to the mnufturer s instrutions. The superntnt ws olleted fter entrifugtion t 12, g for 2 min t 4 C. A formldehyde solution ws used s stndrd. The sorne of stndrd nd smples ws tken t 54 nm using plte reder (Biotek Instruments, In., Vermont, USA). Ctlse tivity ws expressed in nmol/min/mg of protein. Glutthione peroxidse (GSHPx) ws ssyed ording to the

4 4 K. SAHIN ET AL. mnufturer s instrutions (Cymn Chemil, Ann Aror, MI, USA). Liver tissue ws homogenized with the polytron homogenizer in old uffer ( mm Tris- HCL, PH 7.5, 5 mm EDTA, nd 1 mm dithiothreitol) per g tissue nd then entrifuge t 1, g for 15 min t 4 C. This method is sed on the oxidtion of NADPH to NADP +, whih is ompnied y derese in sorne t 34 nm. GSHPx tivity ws mesured y inititing the retion with 2.4 mm umene hydroperoxide. One unit is defined s the mount of enzyme tht oxidizes 1 μmol of NADPH per min t 25 C. The sorne ws red every minute t 34 nm using plte reder (Biotek Instruments, In., Vermont, USA) to otin t lest five time points. The GSHPx tivity ws lulted in nmol/ min/mg of protein. Western lot nlyses For Western lot, protein extrtion ws done y stndrdizing the liver in 1 ml ie-old hypotoni uffer (uffer A), omprising 1 mm HEPES (2-(4-(2-hydroxyethyl)-1-piperzinyl) ethne sulfoni id), ph 7.8, 1 mm KCl, 2 mm MgCl 2, 1 mm dithiothreitol (DTT),.1 mm EDTA, nd.1 mm phenylmethylsulfonyl fluoride (PMSF). The homogente ws mixed with 8 μl of 1% Nonidet P-4 (NP-4) solution nd then entrifuged t 14, g for 2 min. The preipittes were wshed one with μl of Buffer-A plus 4 μl of 1% NP-4, entrifuged nd re-suspended in 2 μl of uffer ontining mm HEPES, ph 7.8, mm KCl, 3 mm NCl,.1 mm EDTA, 1 mm DTT,.1 mm PMSF, nd 2% glyerol), nd re-entrifuged t 14,8 g for 5 min. The superntnt ws olleted nd used for the determintion of NF-κB p65, TNF-α, BCO2, IRS-1, PPAR-γ, Nrf- 2 nd HO-1, ording to the method defined previously [2]. Briefly, μg of proteins were eletrophoresed nd then trnsported onto nitroellulose memrne (Shleiher nd Shuell In., Keene, NH, USA). Antiodies ginst NF-κB p65, TNF-α, BCO2, IRS-1, PPAR-γ, Nrf-2 nd HO-1 (Am, Cmridge, UK) were diluted (1:) in the sme uffer ontining.5% Tween-2. The phosphorylted forms of the ntiodies [nti-nf-kb p65 ntiody (162); nti-ppar gmm ntiody (293) nd nti-nrf2 ntiody (31163)] were used in this study. Protein loding ws ontrolled using monolonl mouse ntiody ginst β- tin (A5316; Sigm). Bnds were exmined densitometrilly using ImgeJ, n imge nlysis system (Ntionl Institute of Helth, Bethesd, USA). Sttistil nlysis All vlues were expressed s men ± SE. The differene etween groups ws evluted y the GLM proedure of SAS (SAS Institute: SAS User s Guide: Sttistis, 22). The tretments were ompred using ANOVA nd Student s unpired t-test, nd P <.5 ws onsidered sttistilly signifint. Results Effet of MZ on metoli helth mrkers HFD used signifint inrese in viserl ft nd liver weight (Tle 2); oth of whih were redued in MZ supplemented rts. The perentge inrese in ody weight of 146.7% in HFD rts ws redued to 139% in MZ supplemented group. There ws lso sustntil inrese registered in the liver weight to ody weight rtio (dt not shown) of HFD-fed rts ompred to ontrols nd MZ-treted groups. MZ supplementtion lso used derese in liver weight. Effet of MZ on iohemil mrkers of rohydrte nd lipid metolism nd the liver nd kidney funtions tests in HFD rts with or without MZ supplementtion There ws signifint rise in gluose, insulin, HOMA-IR, free ftty id nd leptin in HFD-fed rts (Tle 3). MZ supplementtion used onsiderle derese in inresed levels of these sustnes. Tle 2. Effet of MZ supplementtion on the ody weight, viserl ft nd liver weight in rts fed HFD for 12 weeks. Groups Item Control MZ HFD HFD + MZ Initil BW (g) ± ± ± ± 5.74 Finl BW (g) ± 5.65 (122%) ± 6.48 (124.4%) ± 5. (146.7%) ± 8.25 (139%) Feed intke (g/d) ± ± ± ±.78 Viserl ft (g) 7.79 ± ± ± ±.3 Liver (g) ±.21 (4.29%) ±.28 (4.5%) 2.56 ±.6 (6.3%) ±.79 (5.26%) Notes: MZ, mesozexnthin; HFD, high-ft diet; dt re expressed s men±sem of 7 rts from eh group. Supersripts indite tht mens in the sme row without ommon supersript differ signifintly (P <.5).

5 FOOD & NUTRITION RESEARCH 5 Tle 3. Effets of MZ on iohemil prmeter levels in rts fed HFD for 12 weeks. Groups Item Control MZ HFD HFD + MZ Gluose (mmol/l) 4.38 ± ± ± ±.11 Insulin (pmol/l) ± ± ± ± HOMA-IR 8.6 ± ± ± ± 2.84 FFA (mm) 1.23 ± ± ± ±.6 Leptin (ng/ml) 3.71 ± ± ± ± 3.95 Adiponetin (mg/ml) 1.37 ± ± ± ±.2 T-C (mmol/l) 1.32 ± ± ± ±.3 HDL-C (mmol/l).44 ±.1.38 ±.2.54 ±.3. ±.1 LDL-C (mmol/l).69 ±.2.63 ± ±.8.86 ±.1 TG (mmol/l).31 ±.1.27 ±.3.57 ±.3.41 ±.2 AST (U/L) ± ± ± ± 3. ALT (U/L) ± ± ± ± 3.83 ALP (U/L) 212. ± ± ± ± 8.91 Alumin (g/l) 3.35 ± ± ± ±.78 Totl protein (g/l) ± ± ± ±.98 Biliruin (µmol/l) 3.8 ± ± ± ±.21 GGT (U/L) 9.98 ± ± ± ±.2 BUN (mmol/l) 3.55 ± ± ± ±.2 CRE (µmol/l) 7.7 ± ± ± ±.27 Notes: MZ, mesozexnthin; HFD, high-ft diet; FFA, free ftty ids; T-C, totl holesterol; HDL-C, high-density lipoprotein holesterol; LDL-C, low-density lipoprotein holesterol; TG, triglyerides; AST, sprtte minotrnsferse; ALT, lnine trnsferse; ALP, lkline phosphtse; GGT, gmm-glutmyltrnsferse; BUN, lood ure nitrogen; CRE, retinine. Dt re expressed s men±sem of 7 rts from eh group. Supersripts indite tht mens in the sme row without ommon supersript differ signifintly (P <.5). The study reports n lmost % derese in diponetin, nd n inrese in totl holesterol, HDL-C nd LDL-C, s well s TG in HFD rts. The hnges in these iohemil inditors of lipid metolism were rought down to norml y MZ supplementtion. HFD did not use signifint inrese in sprtte trnsminse (AST), lnine trnsminse (ALT), lkline phosphtse (ALP), gmm-glutmyltrnsferse (GGT), ure (BUN) nd retinine (CRE) in the durtion of the tretment, nd the levels remined more or less unffeted in MZ-supplemented rts. Alumin nd totl protein lso remined unffeted in HFD lone or MZ-supplemented nimls fed with HFD. Effet of MZ on the hepti ntioxidnt enzymes nd rotenoid levels As shown in Tle 4, MZ supplementtion used signifint redution in oth serum nd hepti lipid peroxidtion. The serum TAC, liver SOD,CAT nd GSHPx deresed in HFD-fed rts, ut inresed in MZ-supplemented rts, lthough the inrese ws not to the level of ontrol vlues. The tivity of CAT ws lso deresed in HFD rts nd inresed in HFD rts supplemented with MZ. Hepti MZ, Z nd L were lso mesured nd re shown in Tle 2. Mulr rotenoids ould e deteted only in rts dministered with MZ. The hepti onentrtion of MZ, Z, nd L ws higher in the MZ-supplemented group thn in the group fed HFD+MZ diet (P <.1) (Tle 4). Effet of MZ on the level of inflmmtory meditors nd the protein involved in the detoxifition proess MZ redued the elevted level of hepti NF-κB p65 nd TNF-α in HFD-fed rts (Figure 2 Pnels A C). In rts treted with MZ lone, the expression level of these Tle 4. Effets of MZ on the ntioxidnt sttus rts fed HFD for 12 weeks. Groups Item Control MZ HFD HFD + MZ Serum MDA (nmol/ml).87 ±.3.73 ± ± ±.6 Liver MDA (nmol/mg protein) 1.82 ± ± ± ±.12 Serum TAC (nmol Trolox Equiv. per mg protein) 1.18 ± ± ±.5.74 ±.4 Liver SOD (U/mg protein) ± ± ± ± 2.52 Liver CAT (U/mg protein) ± ± ± ± 9.66 Liver GSHPx (U/mg protein) ± ± ± 1.16 d ± 3.54 Zexnthin + meso-zexnthin (mg/g) ± ±.64 Lutein (mg/g) ± ± 1.4 Notes: MZ, mesozexnthin; HFD, high-ft diet; MDA, mlondildehyde; TAC, totl ntioxidnt pity; SOD, superoxide dismutse; CAT, tlse; GSHPx, glutthione peroxidse. Dt re expressed s men±se of 7 rts from eh group. Supersripts indite tht mens in the sme row without ommon supersript differ signifintly (P <.5).

6 6 K. SAHIN ET AL. NFκB, perent of ontrol 2 1 TNFα, perent of ontrol 2 1 BCO2, perent of ontrol 1 d IRS-1, perent of ontrol 1 e d PPAR-γ, perent of ontrol 1 f 1 g 1 h Nrf2, perent of ontrol HO-1, perent of ontrol Figure 2. Hepti NF-κB, TNF-α, BCO2, IRS-1,PPAR-γ Nrf-2 nd HO-1 expression levels in mesozexnthin (MZ) supplemented highft diet (HFD) fed rts nd ontrol groups. Notes: The Western lot strips of the proteins mesured in this study re shown in Pnel A. Pnels B F show the expression level of NF-κB, TNF-α, BCO2, IRS-1, PPAR-γ, Nrf-2 nd HO-1 in vrious groups. The intensity of the nds shown in Pnel A ws quntified y densitometri nlysis. Dt re expressed s rtio of norml ontrol vlue (set to %). Eh r represents the men nd stndrd error of men. Blots were repeted t lest three times (n = 3) nd only representtive lot is shown in Pnel A. β-atin ws inluded to ensure equl protein loding. proteins ws the sme s tht of the ontrol group. When ompred with the untreted HFD group, MZ used signifint inrese in liver BCO2, IRS-1, PPAR-γ (Figure 2 Pnels D F), Nrf-2 nd HO-1 (Figure 2 Pnels G nd H) in HFD rts. MZ supplementtion resulted in ner normliztion of the expression of HO-1 in HFD-fed rts (Figure 2,PnelH). Disussion Prevlene of oesity predisposes to metoli syndrome, whih my led to ftty liver nd hroni onditions inluding T2DM, CVD nd stroke. In moridly oese people, out 9% show histologil defets of the liver nd one-third of these ptients hve ftty ltertion

7 FOOD & NUTRITION RESEARCH 7 involving more thn % heptoytes [6,21]. HFD-indued oesity is reported to impir rohydrte nd lipid metolism [6]. In this study, we oserved signifint inrese in viserl ft nd liver weight in rts fed HFD. The hnges were signifintly rought down in MZ-supplemented nimls, suggesting n overll enefiil effet of MZ on oesity-relted prmeters, thus inditing its possile role in preventing the metoli syndrome. MZ supplementtion lso used signifint derese in gluose, insulin, HOMA-IR, free ftty id nd leptin in HFD fed rts when ompred with the non-supplemented HFD rts. It lso inresed diponetin nd redued holesterol, LDL-C nd TG. There ws no signifint hnge in HDL-C when the MZ-supplemented group ws ompred with HFD-fed rts. Adiponetin is prtiulrly importnt for its role in lipid metolism nd its inrese in MZsupplemented nimls suggests protetive role of diponetin on ftty liver nd hene ssoited omplitions. In this study, MZ tretment did not produe ny sign of toxiity, s indited y the iohemil mrkers of hepti injury. The finding is onsistent with the literture, where MZ is not reported for toxiity [22]. The serum lumin nd totl protein in this study lso remined unffeted. In the urrent study, mg/kg dosge ws hosen on the sis of erlier pulished work where MZ t this dose level showed signifint ntioxidnt effet in rodents [11,16]. With regrd to the effet on hepti mrkers, s reported y Firdous et l. [11], MZ ( nd 2 mg/kg BW) used ner normliztion of the level of hepti mrkers of injury in nitroso diethylmine (NDEA)-indued heptoellulr rinominmlewistrrts.thestudyreportedsignifint inrese in the levels of γ-glutmyl trnspeptidse, lnine minotrnsminse, sprtte minotrnsferse nd lkline phosphtse in NDEA-treted nimls in oth serum nd the liver in dose-dependent mnner, whih ws signifintly rought down y MZ. The study lso reported derese in oxidtive stress mrkers (lipid peroxidtion, onjugted dienes nd tissue hydroperoxides) y MZ nd n inrese in glutthione nd ntioxidnt enzymes (superoxide dismutse, tlse, nd glutthione peroxidse) whih n e ttriuted to the ntioxidnt tivity of mulr rotenoid in HFD fed rts. The effet on oxidtive stress mrkers suggests the protetion of the liver ginst retive oxygen speies generted s result of onsumption of HFD. Erlier, MZ t dose level of mg/kg BW hs een reported y Orhn et l. [16] to protet the retin in HFD fed rts y ounterting the effets of oxidnts in the retin nd regulting growth nd trnsription ftors. Oxidtive stress hs een suggested s usl ftor in the progression of hepti stetosis in nimls [23]. The free rdils nd other retive oxygen speies produed s result of metoli proesses in the ody re ounterted y numer of mehnisms, whih inlude the ntioxidnt enzymes tht onstitute n importnt line of defene ginst these highly retive moieties. The seond line of protetion ginst free rdil dmge is provided y the ntioxidnts. An ntioxidnt is moleule stle enough to donte n eletron to free rdil nd neutrlize its detrimentl result. Vitmins E, C nd rotenoids re some of the ommonly used ntioxidnts. MZ, the mulr rotenoid, hs signifint ntioxidnt tion whih n e used y the onjugted polyene struture of rotenoids. Meso-zexnthin, stereoisomer of zexnthin, hs een reported proly to hve the sme ntioxidnt effet s zexnthin [11]. It hs lso een stted tht in ssoition with zexnthin inding protein, the pi-isoform of glutthione-s-trnsferse, MZ provides etter defene ginst lipidmemrneoxidtionthnzexnthin[24]. The present study suggests tht supplementtion of MZ y its ntioxidnt tivity to HFD-indued oese rts uses redued MDA level nd improved ntioxidnt enzyme (SOD nd CAT) tivities in the liver. Additionlly, we ould not find detetle mount of Z+MZ nd L in the liver of rts tht were not supplemented with MZ. In this study, we stted noteworthy redution in serum nd liver lipid peroxidtion nd n ugmented ntioxidnt response in MZ supplemented nimls, supporting the strong ntioxidnt funtion of MZ in the liver. MZ, euse of its ility to exhiit little or no pro-oxidtive ehviour, even t high rotenoid onentrtion nd t high oxygen tension, hs een suggested to e pure ntioxidnt [25]. Studies pulished erlier hve suggested tht supplementtion of MZ n svenge superoxide nd hydroxyl rdils nd inhiit in vitro lipid peroxidtion [1,11]. It hs lso een reported tht orl dministrtion of MZ inhiited superoxide rdils generted in mrophges y 25.2,.1 nd 67.2% t three different doses of low (), moderte () nd high (2 mg/kg BW) respetively [11]. The present study lso reported n inrese in the tivities of ntioxidnt enzymes like CAT, SOD nd GSHPx in MZ-supplemented rts. These enzymes showed derese in HFD rts ut inresed in MZ supplemented nimls. The results suggest profound ntioxidnt effet of MZ on the liver. The redution in lipid peroxidtion nd the inrese in rotenoids deteted in rts dministered with MZ support the theory tht this rotenoid ought to hve role in lessening the errtions in the liver in treted rts y its ntioxidnt effet [1]. There is no erlier literture relted to investigting the properties of MZ on the liver rotenoid onentrtions in rts fed HFD to ompre with this study. Compounds like gingerol hve erlier een shown to hve nti-oesity tion nd re reported for their enefiil influene on lipid profile, insulin, leptin, mylse nd lipse in oese rts [26]. In linil studies, levels of

8 8 K. SAHIN ET AL. rotenoids, inluding lutein nd zexnthin, re reported to e low in ftty liver [27]. β,β-crotene 9ʹ,1ʹ-oxygense 2 (BCO2), whih leves non-pro-vitmin rotenoids (lutein nd zexnthin) in mitohondri of the liver ells nd is reognized s gtekeeper of mitohondril funtion, hs een reported to mke hepti ells more suseptile to oxidtive stress in mie [28,29]. The ssoition etween rotenoid metolism nd mitohondril iogenesis/mitophgy during the progress of oesity-ssoited hepti stetosis remins unertin [3]. In this study, we found derese in BCO2 in HFD rts. BCO2 inresed in the MZ-supplemented nimls. In reent study [1], MZ hs lso een found to protet the liver nd derese rdio-metoli helth risk s insulin resistne in HFD fed rts. Differenes in insulin level hve reently een reported to e risk ftor of rdio-metoli helth [31], thus impliting MZ not only in dietes ut lso in rdio-metoli helth. Additionl energy onsumption nd deresed energy expenditure hs een reported to stimulte metoli dysfuntion, oxidtive stress nd inflmmtory pthologi ftors suh s IL-6 nd TNF-α [32]. Oxidtive stress nd inflmmtory responses re ruil in the development of type 2 dietes, CVD nd stroke, s well s in liver injury. As disussed ove, CMHR deresed in the MZ supplemented group. MZ signifintly llevited the rdio-metoli helth mrkers nd lso ffeted inflmmtory mrkers in HFD rts. As shown in Figure 2, MZ y virtue of its nti-inflmmtory effet [1] used signifint redution in expression of TNF-α nd NF-κB p65 in HFD-fed rts; in MZ lone treted nimls, the expression level of these proteins ws the sme s tht of the untreted ontrol. On the other hnd, MZ indued signifint inrese in IRS-1, PPAR-γ (Figure 2, E nd F), Nrf-2 nd HO-1 (Figure 2, G nd H), in HFD rts, when ompred with the untreted HFD group. As reported erlier, inresed dipose tissue shows signifint role in the improvement of low-grde inflmmtion, whih is onsidered y ytokine prodution nd stimultion of inflmmtory ytokine signlling pthwys [33]. Pro-inflmmtory ytokines hve een ssoited with dyslipidemi nd theroslerosis [34]. MZ medited derese in pro-inflmmtory ytokines suggests protetive effet of the sustne on the high dipose ondition. IRS-1, the insulin reeptor sustrte 1, signlling dpter protein whih shows signifint role in trnsmitting signls from the insulin nd insulin-like growth ftor-1 reeptors to PI3K/Akt nd Erk MAP kinse pthwys, hs een reported in this study to derese in HFD rts, ut to inrese in the MZ supplemented group. IRS- 1 shows n importnt role in metoli nd mitogeni (growth promoting) pthwys. IRS1 mutnt mie re reported to show pronouned growth impirment ut hd only mild dieti phenotype. Inrese in IRS1 in HFD-fed rts y MZ suggests protetive role in dietes. This study further reported n inrese in PPAR-γ in MZ supplemented HFD rts; PPAR-γ showed signifint derese in HFD rts not treted with MZ. PPAR-γ, whih is lso reognized s the glitzone reeptor, is type II nuler reeptor tht ontrols ftty id storge nd gluose metolism. It stimultes lipid uptke nd dipogenesis y ft ells. PPARG knokout mie hve een reported to fil to generte dipose tissue when fed high-ft diet [35]. Mny nturlly ourring ompounds, inluding vrious polyunsturted ftty ids (PUFA), hve een reported erlier to diretly ind with nd tivte PPAR-γ [36,37]. MZ supplementtion used ner normliztion of the expression of HO-1 in HFD fed rts. It lso inresed the ntioxidnt enzymes tivities (CAT, SOD, nd GSHPx). Heme oxygense (HO) tlyses the degrdtion of heme, thus produing iliverdin, iron nd ron monoxide. There is n induile isoform of HO in response to hevy metls, ytokines nd oxidtive stress, nd n inrese in HO hs een reported to e protetive [38]. An inrese in MZ supplemented HFD fed rts in HO in this study suggests protetive effet of MZ on oxidtive stress. The result is further supported y n inrese in the level of ntioxidnt prmeters nd onomitnt derese in MDA, s reported in this study. The ron monoxide relesed in the retion tlysed y HO n influene vsulr tone independently or influene the funtion of nitri oxide synthse, whih hs n importnt regultory role in metolism. Nutritionl supplementtion hs een found effetive in dietes, nd lso in mintining the norml retinl funtion, mitohondril homeostsis, nd to derese inflmmtory meditors, nd therefore represent n hievle nd inexpensive djunt therpy in dieti ptients [39]. As disussed ove, inresed oxidtive stress nd inflmmtory meditors re implited in the development of dietes nd dieti omplitions, whih n e prevented y ntioxidnts. In dietes, the levels of lutein nd zexnthin hve een reported to derese in the serum [39,4]. Conlusions Tken together, the oservtions in his study led us to onlude tht MZ hs potentil s n djunt therpy to prevent ftty liver nd improve CHR. MZ supplementtion ould exert their helth-enefiting roles in regulting ftty liver y modultion of BCO2, PPAR-γ, IRS-1 NF-κB nd Nrf2 proteins.

9 FOOD & NUTRITION RESEARCH 9 Aknowledgment This study ws sponsored y OmniAtive Helth Tehnologies In. (NJ, USA). This work ws lso supported in prt y the Turkish Ademy of Sienes. Dislosure sttement No potentil onflit of interest ws reported y the uthors. Funding This study ws sponsored y OmniAtive Helth Tehnologies In. (NJ, USA). Referenes [1] Ekel RH, Khn R, Roertson RM, et l. Preventing rdiovsulr disese nd dietes: ll to tion from the Amerin Dietes Assoition nd the Amerin Hert Assoition. Dietes Cre. 26;29: [2] Myns L. Metoli syndrome: insulin resistne nd predietes. FP Essent. 215;435: [3] Pereir MA, Kottke TE, Jordn C, et l. Preventing nd mnging rdiometoli risk: the logi for intervention. Int J Environ Res Puli Helth. 29;6: [4] Wyherley TP, Morn LJ, Clifton PM, et l. Effets of energy-restrited high-protein; low-ft ompred with stndrd-protein; low-ft diets: met-nlysis of rndomized ontrolled trils. Am J Clin Nutr. 212;96: [5] Demir M, Lng S, Steffen HM. Nonloholi ftty liver disese: urrent sttus nd future diretions. J Dig Dis. 215;16: [6] Yki-Järvinen H. Nutritionl modultion of non-loholi ftty liver disese nd insulin resistne. Nutrients. 215;7: [7] Cerf ME. High ft progrmming of et ell ompenstion; exhustion; deth nd dysfuntion. Peditr Dietes. 215;16: [8] Zho L, Zhong S, Qu H, et l. Chroni inflmmtion ggrvtes metoli disorders of hepti ftty ids in high-ft diet-indued oese mie. Si Rep. 215;5:1222. [9] Bone R, Lndrum J, Alvrez-Corre C, et l. Mulr pigment nd serum response to dietry supplementtion with meso-zexnthin. Invest Optom Vis Si. 23;44: [1] Juturu V. Meso-Zexnthin (MZ): urrent perspetives nd new insights. Adv Ophthlmol Vis Syst. 214;4 (1):1. [11] Firdous AP, Preethi KC, Kuttn R. Antioxidnt potentil of meso-zexnthin semi syntheti rotenoid. Food Chem. 21;119: [12] Noln J, Megher K, Kshni S, et l. Wht is mesozexnthin; nd where does it ome from&quest. Eye. 213;27: [13] Firdous AP, Sindhu ER, Kuttn R. Hepto-protetive potentil of rotenoid meso-zexnthin ginst pretmol; CCl4 nd ethnol indued toxiity. Indin J Exp Biol. 211;49:44. [14] Noln JM, Betty S, Megher KA, et l. Verifition of meso-zexnthin in fish. J Food Si Tehnol. 214;5:335. [15] Reeves PG, Nielsen FH, Fhey GC Jr. AIN-93 purified diets for lortory rodents: finl report of the Amerin Institute of Nutrition d ho writing ommittee on the reformultion of the AIN-76A rodent diet. J Nutr. 1993;123: [16] Orhn C, Akdemir F, Tuzu M, et l. Mesozexnthin protets retin from oxidtive stress in rt model. J Oul Phrmol Ther. 216;32: [17] Johnson EJ, Hmmond BR, Yeum KJ, et l. Reltion mong serum nd tissue onentrtions of lutein nd zexnthin nd mulr pigment density. Am J Clin Nutr. 2;71: [18] Krtepe M. Simultneous determintion of sori id nd free mlondildehyde in humn serum y HPLC-UV. LC GC North Ameri. 24;22: [19] Erel O. A novel utomted method to mesure totl ntioxidnt response ginst potent free rdil retions. Clin Biohem. 24;37: [2] Shin K, Tuzu M, Orhn C, et l. Anti-dieti tivity of hromium piolinte nd iotin in rts with type 2 dietes indued y high-ft diet nd streptozotoin. Br J Nutr. 213;11: [21] Clin DJ, Lefkowith JH. Ftty liver disese in morid oesity. Gstroenterol Clin North Am. 1987;16: [22] Thurnhm DI, Howrd AN. Studies on meso-zexnthin for potentil toxiity nd mutgeniity. Food Chem Toxiol. 213;59: [23] Pessyre D. Role of mitohondri in non-loholi ftty liver disese. J Gstroenterol Heptol. 27;22:2 27. [24] Bhosle P, Bernstein PS. Synergisti effets of zexnthin nd its inding protein in the prevention of lipid memrne oxidtion. Biohim Biophys At. 25;174: [25] Mrtin HD, Jäger C, Ruk C. Anti nd prooxidnt properties of rotenoids. J Prkt Chem. 1999;341: [26] Srvnn G, Ponmurugn P, Deep MA, et l. Antioesity tion of gingerol: effet on lipid profile; insulin; leptin; mylse nd lipse in mle oese rts indued y high-ft diet. J Si Food Agri. 214;94: [27] Erhrdt A, Sthl W, Sies H, et l. Plsm levels of vitmin E nd rotenoids re deresed in ptients with Nonloholi Stetoheptitis (NASH). Eur J Med Res. 211;16: [28] Amengul J, Loo GP, Golzk M, et l. A mitohondril enzyme degrdes rotenoids nd protets ginst oxidtive stress. Fse J. 211;25: [29] Morinou T, Tmi H. Bet-rotene 15;15ʹ-dioxygense tivity in streptozotoin-indued dieti rts. J Nutr Si Vitminol (Tokyo). 2;46: [3] Lin C, Rountree CB, Methrtt S, et l. Seondhnd too exposure is ssoited with nonloholi ftty liver disese in hildren. Environ Res. 214;132: [31] Toish B, Bltnizky L, Brki L. Crdiometoli risk ftors nd insulin resistne in oese hildren nd dolesents: reltion to puerty. Peditr Oes. 215;1: [32] Seo DY, Lee S, Figuero A, et l. Aged grli extrt enhnes exerise-medited improvement of metoli prmeters in high ft diet-indued oese rts. Nutr Res Prt. 212;6: [33] Hotmisligil GS, Ery E. Nutrient sensing nd inflmmtion in metoli diseses. Nt Rev Immunol. 28;8:

10 1 K. SAHIN ET AL. [34] Szeknez Z. Pro-inflmmtory ytokines in theroslerosis. Isr Med Asso J. 28;1: [35] Jones JR, Brrik C, Kim KA, et l. Deletion of PPARgmm in dipose tissues of mie protets ginst high ft diet-indued oesity nd insulin resistne. Pro Ntl Ad Si USA. 25;12: [36] Krishnn A, Nir SA, Pilli MR. Biology of PPAR gmm in ner: A ritil review on existing lune. Curr Mol Med. 27;7: [37] Nruhn S, Meissner W, Adhikry T, et l. 15-hydroxyeiostetrenoi id is preferentil peroxisome prolifertor-tivted reeptor et/delt gonist. Mol Phrmol. 21;77: [38] Singh SP, Shrgenheim J, Co J, et l. PGC-1 lph regultes HO-1 expression; mitohondril dynmis nd iogenesis: role of epoxyeiostrienoi id. Prostglndins Other Lipid Medit. 216;125:8 18. pii: S (16)351-X. DOI:1.116/j. prostglndins [39] Kowluru RA, Zhong Q, Sntos JM, et l. Benefiil effets of the nutritionl supplements on the development of dieti retinopthy. Nutr Met (Lond). 214;11:8. [4] Kowluru RA, Menon B, Gierhrt DL. Benefiil effet of zexnthin on retinl metoli normlities in dieti rts. Invest Ophthlmol Vis Si. 28;49: