Food Research & Development, CJ Cheiljedang Corporation, Seoul , Korea; s: (H.-J.K.); (B.S.M.

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1 Int. J. Mol. Si. 214, 15, ; doi:1.339/ijms Artile OPEN ACCESS Interntionl Journl of Moleulr Sienes ISSN The Benefiil Effets of Comined Grpe Pome nd Omij Fruit Extrts on Hyperglyemi, Adiposity nd Hepti Stetosis in d/d Mie: A Comprison with Mjor Index Compounds Su-Jung Cho 1,2, He-Jin Prk 1,2, Un Ju Jung 2, Hye-Jin Kim 3, Byoung Seok Moon 3 nd Myung-Sook Choi 1,2, * 1 Deprtment of Food Siene nd Nutrition, Kyungpook Ntionl University, 137 Snkyuk Dong Puk-ku, Degu 72-71, Kore; E-Mils: hosj1181@nver.om (S.-J.C.); purin613@hotmil.om (H.-J.P.) 2 Center for Food nd Nutritionl Genomis Reserh, Kyungpook Ntionl University, 137 Snkyuk Dong Puk-ku, Degu 72-71, Kore; E-Mil: jungunju@nver.om 3 Food Reserh & Development, CJ Cheiljedng Corportion, Seoul , Kore; E-Mils: hyejink@j.net (H.-J.K.); moons@j.net (B.S.M.) * Author to whom orrespondene should e ddressed; E-Mil: mshoi@knu..kr; Tel.: ; Fx: Externl Editor: Chng Won Choi Reeived: 8 August 214; in revised form: 17 Septemer 214 / Aepted: 18 Septemer 214 / Pulished: 3 Septemer 214 Astrt: This study investigted the effets of omined grpe pome nd omij fruit extrts () on dietes-relted metoli hnges in type 2 dieti d/d mie. The effets of were ompred with those of resvertrol nd shizndrin mixture (), whih is mixture of mjor omponents of. Mie were fed norml diet with (.5% resvertrol nd.2% shizndrin in diet, w/w) or (.3% grpe pome ethnol extrt nd.5% omij fruit ethnol extrt in diet, w/w) for seven weeks. nd not only lowered the levels of lood nd plsm gluose, HA1, insulin nd homeostsis model ssessment of insulin resistne (HOMA-IR) with simultneous derese in hepti gluoneogeni enzymes tivities nd diposity, ut lso improved preservtion of the pnreti β-ells. Plsm leptin nd resistin levels were lower while the plsm diponetin level ws higher in the nd groups thn in the ontrol group. Espeilly, inresed hepti gluokinse tivity nd gene expression nd improved

2 Int. J. Mol. Si. 214, hepti stetosis y elevting ftty id oxidtion ompred to. These findings suggest tht meliortes hyperglyemi, diposity nd hepti stetosis in type 2 dieti mie. Keywords: grpe pome; omij fruit; hyperglyemi; diposity; hepti stetosis 1. Introdution Type 2 dietes is one of the most ommon metoli disorders. The pthogenesis of type 2 dietes is hrterized y omintion of impired insulin seretion from pnreti β-ells nd insulin resistne. In prtiulr, hepti insulin resistne hs een suggested s min ftor in the development of hyperglyemi, euse the liver entrlly ontrols gluose prodution nd utiliztion [1]. Hepti insulin resistne is ssoited with hepti stetosis, whih is highly prevlent in ptients with type 2 dietes [2,3]. Grpes ontin numerous polyphenols, inluding resvertrol, queretin, tehins nd nthoynins, whih hve een shown to redue lood gluose nd improve pnreti β-ell funtion [4]. In grpes, grpe pome ws shown to redue fsting plsm gluose, insulin nd triglyeride levels in growing Sprgue-Dwley rts [5]. As nother lood gluose ontroller, omij (Shisndr hinensis Billon), trditionl Chinese lterntive mediine, hs een used s n nti-dieti gent in Asi [6]. In our previous study, we showed tht omined grpe pome nd omij fruit extrts () meliorted dipogenesis nd hepti stetosis in high-ft diet (HFD)-indued oese mie [7]. We hve investigted here if would not only lower ody ft mss, ut lso improve dietes-relted metoli hnges, inluding plsm iomrkers, β-ell filure nd hepti gluose-regulting enzymes in d/d mie. These mie disply mny of the hrteristis of type 2 dietes, inluding hyperphgi, hyperglyemi, β-ell loss nd insulin resistne. The effets of were ompred with those of resvertrol nd shizndrin mixture (), whih is mixture of the mjor omponents of. Resvertrol is rih in grpes nd is well known to protet ginst oesity, type 2 dietes nd rdiovsulr diseses [8,9]. Shizndrin is the min tive ingredient of omij nd hs een shown to lower lood gluose level nd improve insulin resistne in 9% pnretetomized dieti rts fed n HFD [1]. 2. Results nd Disussion We found tht signifintly lowered the levels of lood nd plsm gluose nd HA1 nd deresed the homeostsis model ssessment of insulin resistne (HOMA-IR) vlue, similrly to or etter thn supplementtion (Figure 1A C). Interestingly, hepti gluose-6-phosphtse (G6Pse) nd phosphoenolpyruvte roxykinse (PEPCK) tivities were mrkedly downregulted in - nd -treted groups thn in the ontrol, nd ws even more effetive thn in inhiiting hepti G6Pse nd PEPCK tivities in the liver (Figure 1D). Moreover, nd tretments signifintly inresed hepti gluokinse (GK) tivity more thn the ontrol (CON) group (Figure 1D). This is onsistent with their greter gluose-lowering effets (Figure 1A). nd tretments lso signifintly inresed hepti GK mrna expression nd deresed hepti G6Pse mrna

3 Int. J. Mol. Si. 214, expression thn the ontrol (Figure 1E). Hepti G6Pse nd PEPCK tivities re elevted in type 2 dieti nimls, suh s d/d mie, when ompred to norml nimls [11]. However, GK, key glyolyti enzyme, is downregulted in d/d mie [12]. Do et l. [9] indited tht resvertrol led to lter dietes-relted metolism in d/d mie tht inludes the glyemi ontrol effet of grpes. The nd tretments in this present study ffeted gluose metolism diretly without ltering ody weights. Insulin, whih is sereted y pnreti β-ells, is signifintly elevted in insulin resistne nd/or deresed β-ell mss to mintin gluose homeostsis [13]. The d/d mie exhiit hyperinsulinemi to ompenste for inresed insulin resistne, nd hyperglyemi mnifests t four to eight weeks of ge, following β-ell filure [14]. In this study, nd tretments signifintly deresed plsm insulin nd the insulin/glugon rtio in ddition to preserving pnreti β-ells ompred to the ontrol (Figure 1F,G). Along with the solute onentrtion of plsm insulin, the plsm insulin/glugon rtio is ssoited with the regultion of hepti gluose metolism, euse glugon stimultes gluoneogenesis nd glyogenolysis [15]. We oserved tht preserved islet rhiteture nd inresed the pnreti insulin-stined re in d/d mie, nd the protetive effet on pnreti β-ells ws more evident in the group thn in the group (Figure 1G). Woods et l. [16] hve shown tht plsm insulin level positively orreltes with diposity, espeilly viserl diposity. Adipokines, whih re sereted from dipose tissue nd inlude leptin, resistin nd diponetin, hve lso een proposed s link mong oesity, insulin resistne nd dietes [17]. Leptin positively orreltes with ody mss index, downregultes β-ell expression of interleukin (IL)-1 reeptor ntgonist nd inreses the relese of IL-1β in humn islets, leding to impired β-ell funtion nd β-ell deth [18]. Resistin indues insulin resistne in islet β-ells, impirs gluose-indued insulin seretion nd positively orreltes with white dipose tissue (WAT) mss [18]. Adiponetin stimultes gluose utiliztion nd ftty id oxidtion, therey diretly regulting gluose metolism nd insulin sensitivity. In this study, onsistent with the plsm insulin levels, the totl WAT weights were signifintly deresed in - nd -treted groups thn in the ontrol (Figures 1F nd 2A). The - nd -supplemented mie lso hd smller epididyml dipoytes thn mie in the ontrol group (Figure 2B). In ddition, nd signifintly deresed the plsm leptin nd resistin levels, ut inresed plsm diponetin levels ompred to the ontrol (Figure 2C E). Our previous study showed tht grpe skin extrt meliorted hepti stetosis through derese in hepti lipogeni enzyme tivity onomitnt with β-oxidtion tivtion in HFD-indued oese mie, nd shizndrin deresed hepti holesterol nd triglyeride levels in hyperholesterolemi mie [19,2]. In this study, the nd groups deresed the numer nd size of lipid droplets in the liver of d/d mie thn the ontrol (Figure 2F). Furthermore, supplementtion with, s well s not only mrkedly deresed the tivities of enzymes for de novo ftty id synthesis, ftty id synthse (FAS) nd gluose-6-phosphte dehydrogense (G6PD), ut lso inresed ftty id oxidtion in the liver ompred to the ontrol (Figure 2G). FAS tlyzes the synthesis of sturted long-hin ftty id from etyl-coa, mlonyl-coa nd NADPH, nd G6PD is involved in supplying NADPH for ftty id iosynthesis. Thus, the redution of hepti FAS nd G6PD tivities n limit the vilility of long-hin ftty ids tht re required for hepti triglyeride synthesis. Hepti ftty id oxidtion lso diminishes hepti triglyeride synthesis. On the other hnd, irulting lipids n e delivered to the liver nd re relted to the tivities of hepti lipid-regulting enzymes [21]. The

4 G6Pse (umol/min/mg protein) PEPCK (umol/min/mg protein) GK (nmole/min/mg protein) Reltive mrna ritrry unit Blood gluose (mg/dl) HA 1 (%) Int. J. Mol. Si. 214, nd tretments lowered plsm free ftty id (FFA) levels more thn the untreted ontrol, nd lso lowered plsm triglyeride nd totl-holesterol levels ompred to the ontrol (Figure 3). It is plusile tht synergisti effets my exist etween totl flvonoids nd polyphenols of extrts of grpe pome nd omij in protetive effets on hyperglyemi, diposity nd hepti stetosis in type 2 dieti mie. Other unknown tive omponents present in extrts of grpe pome nd omij ould lso ply importnt roles in their iologil effets. Figure 1. Effets of nd on (A) lood gluose; (B) plsm gluose nd HOMA-IR; (C) lood HA1; (D) hepti enzymes tivities; (E) hepti genes expression; (F) plsm insulin nd insulin/glugon rtio nd (G) immunohistohemistry stining for pnreti insulin in C57BL/KsJ-d/d mie. A F: Dt re the mens ± SE (n = 1).,, Mens not shring ommon letter re signifintly different mong the groups t p <.5; G: Representtive photomirogrphs of the pnres re shown t 2 mgnifition (n = 1). Arrow, β-ell. CON, norml diet ontrol;, norml diet plus resvertrol (.5%, w/w) nd shizndrin (.2%, w/w);, norml diet plus grpe pome extrt (.3%, w/w) omined with omij fruit extrt (.5%, w/w); HOMA-IR, homeostti model ssessment-insulin resistne = [fsting insulin onentrtion (mu/l)] [fsting gluose onentrtion (mg/dl).5551]/22.5; G6Pse, gluose-6-phosphtse; PEPCK, phosphoenolpyruvte roxykinse; GK, gluokinse. D A 6 B 9 C CON 5 3 CON Initil Finl Plsm gluose HOMA-IR (mg/dl) F CON CON Insulin (ng/ml) Hepti Enzymes Ativities CON Insulin(pg/mL)/ Glugon(pg/mL) G CON CON E CON CON Pnreti Insulin Immunohistohemistry Hepti Genes Expression G6Pse PEPCK GK

5 FAS (nmol/min/mg protein) G6PD (nmol/min/mg protein) β-oxidtion (nmol/min/mg protein) ritrry unit) Leptin (ng/ml) Resistin (ng/ml) Adiponetin (μg/ml) Totl WAT weight (g/1g B.W.) Int. J. Mol. Si. 214, Figure 2. Effets of nd on (A) totl WAT weight; (B) epididyml WAT morphology; (C) plsm leptin; (D) resistin; (E) diponetin; (F) liver morphology; (G) hepti enzymes tivities nd (H) gene expression in C57BL/KsJ-d/d mie. A,C E,G,H: Dt re the mens ± SE (n = 1).,, Mens not shring ommon letter re signifintly different mong the groups t p <.5; B,F: Representtive photomirogrphs of epididyml WAT nd liver re shown t 2 mgnifition (n = 1). Arrows, dipoytes (B) nd lipid droplets (F). CON, norml diet ontrol;, norml diet plus resvertrol (.5%, w/w) nd shizndrin (.2%, w/w);, norml diet plus grpe pome extrt (.3%, w/w) omined with omij fruit extrt (.5%, w/w); WAT, white dipose tissue; FAS, ftty id synthse; G6PD, gluose-6-phosphte dehydrogense. A 21 B Epididyml WAT Morphology CON CON C D E 6 4 F Liver Morphology CON CON CON CON G Hepti Enzymes Ativities H Hepti Gene Expression CON CON CON FAS (reltive mrna CON

6 Plsm lipids (mmol/l) Int. J. Mol. Si. 214, Figure 3. Effets of nd on plsm lipids levels in C57BL/KsJ-d/d mie. Dt re the mens ± SE (n = 1)., Mens not shring ommon letter re signifintly different mong the groups t p <.5. CON, norml diet ontrol;, norml diet plus resvertrol (.5%, w/w) nd shizndrin (.2%, w/w);, norml diet plus grpe pome extrt (.3%, w/w) omined with omij fruit extrt (.5%, w/w) CON Free ftty id Triglyeride Totl holesterol 3. Experimentl Setion 3.1. Mterils Grpes (Vitis vinifer L., MBA (Must iley A) speies) nd omij (Shisndr hinensis Billon) were purhsed from Gyeongsnguk-do, Kore. Resvertrol nd shizndrin were purhsed from Sigm Chemil Corportion Preprtion of Extrts In this study, grpe pome (skin nd stem) nd omij fruits (Frutus Shisndre) were used. Smples were prepred y dding 2 L of 8% nd 5% ethnol to 1 g of dried grpe pome nd omij fruit, respetively; extrtion ws done t 8 C for 2 h nd then ooled. The solution ws filtered (Whtmn pper No. 2), onentrted with rotry vuum evportor nd stored t 7 C. The finl weight of the grpe pome ethnol extrt ws 19.9 g (reovery rte: 19.9%) nd of the omij fruit ethnol extrt ws 39.7 g (reovery rte: 39.7%), s shown in Supplementl Tle S1. Resvertrol nd shizndrin re eh representtive index ompounds present in grpe pome nd omij, respetively. As shown in Supplementl Tle S1, 1 g of grpe pome ethnol extrt ontins.2 mg of resvertrol, 52 mg of totl flvonoid nd 95 mg of totl polyphenol. One grm of omij fruit ethnol extrt ontins 8 mg of shizndrin, 7 mg of totl flvonoid nd 32 mg of totl polyphenol.

7 Int. J. Mol. Si. 214, HPLC Anlysis of Index Compounds Index hemil onentrtions were simultneously determined using the HPLC method. The pprtus used ws n Agilent 11/12 system (Agilent, Wldronn, Germny) onsisting of inry pump, thermostted utosmpler (injetion volume = 3 μl nd 2 μl, grpe pome ethnol extrt nd omij fruit ethnol extrt, respetively), olumn oven (set = 35 C) nd DAD detetor (36 nd 254 nm, grpe pome ethnol extrt nd omij fruit ethnol extrt, respetively). The olumn ws n Elipse XDB-C18, 25 mm 4.6 mm, 5 μm (Agilent). Eluent ws etonitrile nd wter with.5% (v/v) TFA grdient. Flow rtes were.6 nd.8 ml/min, nd running times were 25 min, for grpe pome ethnol extrt nd omij fruit ethnol extrt, respetively. The HPLC hromtogrms of shizndrin stndrd, omij extrt, resvertrol stndrd nd grpe pome extrt re illustrted in Supplementl Figure S1C, S1D, S2C nd S2D Animl nd Diets Mle C57BL/KsJ-d/d mie were purhsed from the Jkson Lortory (Br Hror, ME, USA) t 4 weeks of ge. The nimls were individully housed t onstnt temperture (24 C), 12 h light/drk yle nd fed pelletized ommeril non-purified diet for 1 week fter rrivl. The mie were then rndomly divided into 3 groups (n = 1) nd fed the respetive experimentl diets for 7 weeks: norml ontrol diet, resvertrol nd shizndrin diet (,.5% resvertrol nd.2% shizndrin with CON diet, w/w) nd the omined extrts of grpe pome nd omij fruit diet (,.3% grpe pome extrt nd.5% omij fruit extrt with CON diet, w/w). The omposition of the diets is presented in Tle 1. The mie hd d liitum ess to food nd distilled wter during the experimentl period. Their food intke nd ody weight were mesured dily nd weekly, respetively. On the 7th week, mie were nesthetized with diethyl ether nd srified fter 12 h of fsting. Blood ws tken from the inferior ven v nd then entrifuged t 1 g for 15 min t 4 C, nd the plsm ws seprted to nlyze the plsm iomrkers. After lood olletion, the liver nd dipose tissues were promptly removed, rinsed, weighed, frozen in liquid nitrogen nd stored t 7 C. The pnres ws lso removed, rinsed nd fixed in 1% hydrogen peroxide. This niml study protool ws pproved y the Ethis Committee for niml studies t Kyungpook Ntionl University, Repuli of Kore Plsm nd Blood Biomrkers The levels of plsm insulin, glugon, leptin, resistin nd diponetin were determined with multiplex detetion kit from Bio-Rd (Herules, CA, USA). All smples were ssyed in duplite nd nlyzed with Luminex 2 Lmp system (Luminex, Austin. TX, USA). Dt nlyses were done with the Bio-Plex Mnger softwre version (Bio-Rd, Herules, CA, USA). The lood gluose onentrtion ws mesured with whole lood otined from the til veins fter withholding food for 12 h using gluose nlyzer, GluDr supersensor (Allmedius, Kore). The lood glyosylted hemogloin (HA1) onentrtion ws mesured with n nlyzer (MiormtTM I Hemogloin A1 Test, Bio-Rd, CA), nd the plsm gluose level ws nlyzed with ommerilly ville kit (Asn, Seoul, Kore). The HOMA-IR ws lulted s previously desried: HOMA-IR = [fsting insulin onentrtion (mu/l)] [fsting gluose onentrtion (mg/dl).5551]/22.5 [22]. Plsm lipid

8 Int. J. Mol. Si. 214, onentrtions were determined with ommerilly ville kits: totl-holesterol, triglyeride (Asn, Seoul, Kore) nd FFA (Wko Chemils, Rihmond, VA, USA). Tle 1. Composition of experimentl diets (unit: % of diet). CON, norml diet ontrol;, norml diet plus resvertrol (.5%, w/w) nd shizndrin (.2%, w/w);, norml diet plus grpe pome extrt (.3%, w/w) omined with omij fruit extrt (.5%, w/w). Ingredients CON Csein D,L-Methionine Surose Cellulose AIN-minerl AIN-vitmin Choline itrtrte Corn Strh Corn oil tert-butylhydroquinone Resvertrol.5 Shizndrin.2 Grpe pome extrt.3 Omij fruit extrt.5 Totl AIN-76 minerl mixture (grms/kg): lium phosphte 5, sodium hloride 74, potssium itrte 222, potssium sulfte 52, mgnesium oxide 24, mngnous ronte 3.5, ferri itrte 6, zin ronte 1.6, upri ronte.3, potssium iodte.1, sodium elenite.1, hromium potssium sulfte.55, surose 118.3; 2 AIN-76 vitmin mixture (grms/kg): thimin HCl.6, rioflvin.6, pyridoxine HCl.7, niin 3, lium pntothente 1.6, foli id.2, iotin.2, vitmin B 12 1, vitmin A (5, U/gm).8, vitmin D 3 (4, U/gm).25, vitmin E ette (5 U/gm) 1, mendione sodium isulfite.8, surose Hepti Enzymes Ativities Enzyme soures were prepred ording to the method developed y Hulher nd Oleson with slight modifition [23]. The GK tivity ws determined from liver smples homogenized in 9 volumes of uffer ontining 5 mmol/l Tris-HCl, ph 7.4, 1 mmol/l KCl, 1 mmol/l merptoethnol nd 1 mmol/l EDTA. Homogentes were entrifuged t 1, g for 1 h; the ytosol ws used for the spetrophotometri ssy s desried y Dvidson nd Arion, in whih the formtion of gluose-6-phosphte from gluose t 37 C ws oupled to its oxidtion y G6PD nd niotinmide denine dinuleotide (NAD) [24]. The G6Pse tivity ws determined in the mirosome with spetrophotometri ssy ording to the method y Alegre et l. [25]. The retion mixture ontined the following: 1 mmol/l sodium Hepes (ph 6.5), 26.5 mmol/l gluose-6-phosphtte nd 1.8 mmol/l EDTA, oth previously djusted to ph 6.5, 2 mmol/l NADP +,.6 IU/L mutrotse nd 6 IU/L gluose dehydrogense. The PEPCK tivity ws determined ording to the method desried y Bentle nd Lrdy [26]. The retion mixture ontined the following in 1-mL finl volume: 77 mol/l sodium Hepes, 1 mmol/l IDP, 1 mmol/l MnCl2, 1 mmol/l dithiothreitol,.25 mmol/l NADH, 2 mmol/l phosphoenolpyruvte, 5 mmol/l NHCO3 nd 7.2 U of mli

9 Int. J. Mol. Si. 214, dehydrogense. The mount of protein in the enzyme soures ws determined with the Brdford method using ovine serum lumin s the stndrd [27]. Ftty id β-oxidtion tivity ws mesured spetrophotometrilly y monitoring the redution of NAD to NADH in the presene of plmitoyl-coa, s desried y Lzrow [28]. The results were expressed s nmol/min per mg of protein. FAS tivity ws determined ording to the method desried y Crl et l. through monitoring the mlonyl-coa-dependent oxidtion of NADPH t 34 nm, in whih the tivity represents the oxidized NADPH nmol/min per mg of protein [29]. The G6PD tivity ws ssyed y spetrophotometri methods ording to the proedures desried y Pitkänen et l., in whih the tivity ws expressed s the redued NADPH nmol/min per mg of protein [3] RNA Extrtion nd Rel-Time Quntittive PCR Anlysis Totl RNA ws isolted from the liver using TRIzol regent (Invitrogen Life Tehnologies, Grnd Islnd, NY) ording to the mnufturer s instrutions. DNse digestion ws used to remove ny DNA ontmintion, nd RNA ws re-preipitted in ethnol to ensure no phenol ontmintion. The RNA purity nd integrity were evluted with the Agilent 21 Bionlyzer (Agilent Tehnologies, Plo Alto, CA, USA). Equl mounts of RNA from eh experimentl group were pooled to normlize individul differenes. Totl RNA (1 μg) ws reverse trnsried into DNA using the QuntiTet reverse trnsription kit (Qigen, Berlin, Germny). Then, mrna expression ws quntified y rel-time quntittive PCR, using the QuntiTets SYBR green PCR kit (Qigen, Germny) on the CFX96TM rel-time PCR system (Bio-Rd, UK). The sequenes of the primers were s follows: FAS, 5'-CGCTCCTCGCTTGTCGTCTG-3' (forwrd), 5'-AGCCTTCCATC-TCCTGTCATC ATC-3' (reverse); G6Pse, 5'-GGAGGAAGGATGGAGGAAGGAATG-3' (forwrd), 5'-GGTCAGCA ATCACAGACACAAGG-3' (reverse); GAPDH (glyerldehyde-3-phosphte dehydrogense), 5'-ACAATGAATACGGCTACAGCAACAG-3' (forwrd), 5'-GGTGGTCCAGGGTTTCTTACTCC-3' (reverse); GK, 5'-CAGGACAGTGGAGCGTGAAGAC-3' (forwrd), 5'-TTACAGGGAAGGAGA AGGTGAAGC-3' (reverse); PEPCK, 5'-GGAGGAAGGATGGAGGAAGGAATG-3' (forwrd), 5'-GGTCAGCAATCACAGACACAAGG-3' (reverse). Cyle thresholds were determined sed on the SYBR green emission intensity during the exponentil phse. The fold hnges were lulted using the 2 Ct method; trnsripts of GAPDH were lso mplified from the smples in order to vlidte the internl ontrol genes for rel-time quntittive PCR detetion Histopthologil Anlysis Liver nd epididyml ft were removed nd fixed in uffer solution of 1% formlin. Fixed tissues were proessed routinely for prffin emedding, nd 4-μm setions were prepred nd dyed with hemtoxylin-eosin. Stined res were viewed using n optil mirosope with mgnifying power of 2. For the immunohistohemistry of pnreti β-ells, the islet ws setioned, fixed in 1% hydrogen peroxide nd wshed in.1 M itrte uffer (ph 6.). These setions were treted with loking regent (Ultr Teh HRP) to prevent nonspeifi inding nd inuted with monolonl ntiodies ginst insulin (Snt Cruz Bioteh, In., Snt Cruz, CA, USA). Antiody retivity ws deteted using HRP-onjugted iotin-streptvidin omplexes nd developed with diminoenzidine

10 Int. J. Mol. Si. 214, tetrhydrohloride (DAB) s the sustrte. Stined res were viewed using n optil mirosope with mgnifying power of Sttistil Anlysis The sttistil nlyses were performed with the sttistil pkge for soil siene softwre progrm (SPSS, In., Chigo, IL, USA). Signifint differenes etween the mens were determined y one-wy ANOVA. Dunn s multiple-rnge test ws performed if differenes were identified etween the groups t p <.5. All dt re expressed s the men with its stndrd error of the men. 4. Conlusions Our findings lerly indite tht the supplementtion of meliortes lood nd plsm gluose levels, HA1 level, HOMA-IR vlue, diposity nd hepti stetosis in type 2 dieti d/d mie, t lest in prt, through the preservtion of β-ell insulin expression nd hepti insulin sensitivity. Aordingly, the positive metoli effets of dietry ould e prtly medited y deresing gluose nd ftty id synthesis nd/or inresing their utiliztion in the liver y regulting enzyme tivities involved in glyolysis, gluoneogenesis, ftty id synthesis nd oxidtion (Supplementl Figure S3). Moreover, enefiil metoli effets re relted to improved dipokine prodution. Further studies re needed to determine the optiml rtios of grpe pome nd omij s dietry supplement in humn sujets with high lood gluose. Supplementry Mterils Supplementry mterils n e found t Aknowledgments This work ws supported y the High Vlue-dded Food Tehnology Development Progrm (No ), the Ministry for Food, Agriulture, Forestry nd Fisheries, y the Ntionl Reserh Foundtion of Kore grnt (No , NRF-212M3A9C448818) nd y the Center for Women In Siene, Engineering nd Tehnology (WISET) Grnt funded y the Ministry of Siene, ICT nd Future Plnning of Kore (MSIP) under the Progrm for Returners into R&D, Repuli of Kore. Author Contriutions Su-Jung Cho: performed the experiments nd wrote the mnusript. He-Jin Prk: ssisted the first uthor in performing the experiments. Un Ju Jung: offered ssistne to the orresponding uthor in the mnusript writing nd its English-lnguge editing. Hye-Jin Kim nd Byoung Seok Moon: designed the study nd prepred the extrts. Myung-Sook Choi: responsile for designing the study, summrizing the results nd writing the mnusript. Conflits of Interest The uthors delre no onflit of interest.

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