Curcumin attenuates Nrf2 signaling defect, oxidative stress in muscle and glucose intolerance in high fat diet-fed mice

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1 Online Sumissions: fice doi:239/wjd.v3.i.94 World J Dietes 212 My 1; 3(): ISSN (online) 212 Bishideng. All rights reserved. ORIGINAL ARTICLE Curcumin ttenutes Nrf2 signling defect, oxidtive stress in muscle nd glucose intolernce in high ft diet-fed mice Hui-Jun He, Guo-Yu Wng, Yun Go, Wen-Hu Ling, Zhi-Wen Yu, Tin-Ru Jin Hui-Jun He, Guo-Yu Wng, Yun Go, Wen-Hu Ling, Zhi- Wen Yu, Tin-Ru Jin, Deprtment of Nutrition, School of Pulic Helth, Sun Yt-sen University, Room 17, 74 Zhongshn 2 nd Rod, Gungzhou 18, Gungdong Province, Chin Author contriutions: He HJ performed the experiments throughout the study nd wrote the drft of this mnuscript; Wng GY nd Go Y prticipted in the intrperitonel glucose tolernce test experiments nd mice tissue collection; Ling WH provided his dvice on feeding nd the dosge of curcumin in this study; Yu ZW designed the study, provided technicl guidnce nd edited this pper; Jin TR coordinted the study, provided importnt guidnce nd finl editing of the mnuscript. Supported y A Ntionl Nturl Science Foundtion of Chin Grnt, No to Jin TR nd Yu ZW Correspondence to: Dr. Zhi-Wen Yu, Associte Professor, Deprtment of Nutrition, School of Pulic Helth, Sun Yt-sen University, Room 17, 74 Zhongshn 2 nd Rod, Gungzhou 18, Gungdong Province, Chin. yuzhiwen@yhoo.com Telephone: Fx: Received: Jnury 29, 212 Revised: April 19, 212 Accepted: My 11, 212 Pulished online: My 1, 212 Astrct AIM: To investigte the signling mechnism of ntioxidtive ction y curcumin nd its impct on glucose disposl. METHODS: Mle C7BL/6J mice were fed with either norml diet (n = 1) or high ft diet () (n = 2) to induce oesity nd insulin resistnce. After 16 wk, 1 -fed mice were further treted with dily curcumin orl gvge t the dose of mg/kg ody weight (BW) ( + curcumin group). After 1 d of the curcumin supplementtion, n intrperitonel glucose tolernce test ws performed. Fsting lood smples were lso collected for insulin nd glucose mesurements. Insulin-sensitive tissues, including muscle, dipose tissue nd the liver, were isolted for the ssessments of mlondildehyde (MDA), rective oxygen species (ROS) nd nucler fctor erythroid-2-relted fctor-2 (Nrf2) signling. RESULTS: We show here tht in mouse model, short-term curcumin gvge ttenuted glucose intolernce without ffecting -induced BW gin. Curcumin lso ttenuted -induced elevtions of MDA nd ROS in the skeletl muscle, prticulrly in its mitochondril frction, ut it hd no such n effect in either dipose tissue or the liver of -fed mice. Correspondingly, in skeletl muscle, the levels of totl or nucler content of Nrf2, s well s its downstrem trget, heme oxygense-1, were reduced y -feeding. Curcumin intervention drmticlly reversed these defects in Nrf2 signling. Further nlysis of the reltionship of oxidtive stress with glucose level y regression nlysis showed positive nd signi ficnt correltion etween the re under the curve of glucose tolernce test with MDA levels either in muscle or musculr mitochondri. CONCLUSION: These findings suggest tht the shortterm tretment of curcumin in -fed mice effectively meliortes musculr oxidtive stress y ctivting Nrf2 function tht is novel mechnism for its effect in improving glucose intolernce. 212 Bishideng. All rights reserved. Key words: Oxidtive stress; Insulin resistnce; Glucose tolernce; Nucler fctor erythroid-2-relted fctor-2; Curcumin; Mitochondri Peer reviewers: Suresh T Mthews, Professor, Deprtment of Nutrition nd Food Science, Auurn University, Auurn, AL 36849, United Sttes;; Dr. Serp Ylin, Phrmcy Fculty, Mersin University, Mersin 33169, Turkey He HJ, Wng GY, Go Y, Ling WH, Yu ZW, Jin TR. Curcumin ttenutes Nrf2 signling defect, oxidtive stress in muscle nd glucose intolernce in high ft diet-fed mice. World J Dietes 94 My 1, 212 Volume 3 Issue

2 He HJ et l. Curcumin ctivtes Nrf2 nd ttenutes insulin resistnce 212;; 3(): Aville from: URL: wjgnet.com/ /full/v3/i/94.htm DOI: org/239/wjd.v3.i.94 INTRODUCTION Oesity is ssocited with high incidence of mny metolic disorders, including type 2 dietes mellitus nd crdiovsculr diseses [1,2]. Insulin resistnce is recognized s core mechnism for these oesity-relted diseses. Extensive recent studies hve shown tht chronic ctivtion of inflmmtory signling, endoplsmic reticulum (ER) stress nd mitochondril dysfunction remongthe mong etiologiclicl fctors of insulinresistnce [3,4]. In ddition, the cusl reltionship etween oxidtive stress nd the development of insulin resistnce [-7] hs een suggested for long time, while ithsginedmuchmorettention more ttention recently due to the recognitionof of theinvolvementof of mitochondril-derived oxidtive stress in the etiology of insulin resistnce [4,8]. Over nutrition supply or high ft diet () consumption led to incresed mitochondril oxidtive phosphoryltion nd inevitle rective oxygen species (ROS) )over-production [7,8]. Mmmls however, hve evolved endogenous nti-oxidtive systems to prevent oxidtive stress nd mintin insulin sensitivity. The trnscription nucler fctor erythroid-2-relted fctor-2 (Nrf2) plys mjor role in mintining redox lnce. In resting cell, Nrf2 molecules minly reside in cell cytosol nd re nchored with its negtive regultor Kelch-like ECH-ssocited protein 1 (Kep1). The coupling etween Nrf2 nd Kep1 leds to Nrf2 protesoml degrdtion [9]. When oxidtive stress occurs, Nrf2 nd Kep1 will e seprted, leding to the elevtion of free Nrf2 level nd the increse of Nrf2 nucler trnsloction. Nucler Nrf2 will then ind to the consensus nucleotide sequence, nmely ntioxidnt response element (ARE), in the promoter regions of ttery of genes tht encode ntioxidnt enzymes [9,1]. Since oxidtive stress is n importnt etiologicl fctor in ging, inflmmtion nd tumor growth, it is not surprising tht Nrf2 cn modulte the progress of these diseses [11,12]. Interestingly, certin nturl phyto-compounds, including curcumin, re Nrf2 ctivtors nd hve potentil therpeutic effect for diseses, including inflmmtion, tumor, hert filure, neurodegenertive diseses nd ischemi [13-16]. Curcumin hs lso een shown to sensitize insulin ction in -fed dietic models [17]. The understnding of moleculr mechnisms underlying the insulin sensitizing ction y curcumin is essentil for the genertion of interventionl pproches. Most in vivo investigtions hve een conducted to explore long-term effect of curcumin nd its eneficil effect on insulin signling minly vi reducing ody weight (BW) gin nd inhiiting inflmmtory rections [17,18]. However, in spite of the documented nti-oxidtive function of curcumin [14], no ttempt hs een mde to correlte the role of curcuminmedited nti-oxidtive function nd its impct on insulin sensitivity. Since curcumin hs een shown to ctivte Nrf2 in cultured cells [1], here we sked whether curcumin ctivtes Nrf2 system in vivo nd whether the ctivtion leds to improved insulin signling. To investigte whether the eneficil effect of curcumin cn e chieved independent of its BW lowering effect, we delivered low dose of curcumin y gvge short-term to void BW chnge. MATERIALS A METHODS Mterils Mle C7BL/6J mice (8 wk of ge) were purchsed from the Lortory Animl Center of Sun Yt-sen University. Curcumin (curcuminoid content 94%), common chemicls nd protese inhiitors were otined from Sigm Chemicl Compny (St Louis, MO). Norml diet () (8% clories from ft) nd (6% clories from ft) were provided y Gungdong Animl Center, (Gungzhou, Gungdong, Chin). Nrf2 ntiody ws from Snt Cruze Biotechnology (Snt Cruz, CA). Heme oxygense-1 (HO-1) nd -ctin ntiodies were from the Proteintech Group (Chicgo, IL). Histone H3 ntiody ws otined from Cell Signling Technology (Denvers, MA, United Sttes). Bicinchoninic cid (BCA) ssy kit ws purchsed from Pierce Bio-technology, Inc. (Rockford, IL). Enhnced chemiluminescence (ECL) ws purchsed from Thermo Scientific (Rockford, IL). Blood glucose meter ws otined from ACON Lortories, Inc. (Sn Diego, CA). ROS ssy kit ws purchsed from GENMED Scientifics (Shnghi, Chin). Mlondildehyde (MDA) ssy kit ws purchsed from ZeptoMetrix (Bufflo, NY). Animl cre nd tretment The niml experiments were performed in ccordnce with the Guide for Cre nd Use of Experimentl Animls (Sun Yt-sen University, SYSU). Thirty mice were housed in n environmentlly controlled room t 22 ± 2. nd % ± % humidity with 12-h: 12-h light/drk cycle. The mice hd ccess to food nd wter d liitum. After one week dptive period, the mice were rndomly divided into two weight-mtched groups. Ten mice were fed with nd the rest of them (2 mice) were fed with. BW ws mesured weekly. After 16 wk of feeding, -fed mice were further divided into two weight-mtched groups, e.g., group (n = 1) nd plus curcumin treted group (n = 1). Curcumin ws given dily y orl gvge t the dose of mg/kg BW in 1% croxymethyl cellulose uffer solution for 1 d. Mice in the nd group were gvged with vehicle only. For lood smple nd tissue collection, ll mice were euthnized fter fsting for 6 h. Blood smples were tken nd centrifuged t 4 t 3 rpm for 1 min for collecting the serum. Menwhile, skeletl muscle in qudriceps, livers nd epididyml ft pds were rpidly isolted, followed y immedite freezing in liquid nitrogen nd then stored t -8 efore further nlyses. 9 My 1, 212 Volume 3 Issue

3 He HJ et l. Curcumin ctivtes Nrf2 nd ttenutes insulin resistnce Intrperitonel glucose tolernce test Intrperitonel glucose tolernce test ws performed s previously descried [19]. Briefly, mice were fsted overnight, followed y glucose (1 g/kg) injection intrperitonelly. Blood smples collected from til vein were used for glucose mesurement. Mitochondril nd nucler frctiontion Nucler nd mitochondril frctiontion were performed s previously descried [2,21] with slight modifiction. Aout 3 mg skeletl muscle ws homogenized in 1 ml ice-cold uffer contining 2 mmol/l HEPES (ph 7.4), 2 mmol/l sucrose, 1 mmol/l KCl, 1. mmol/l MgCl2, 1 mmol/l EDTA, 1 mmol/l EGTA, 1 mmol/l dithiothreitol nd the protese inhiitors [2 μg/ml protinin, μg/ml leupeptin nd 2 mmol/l phenylmethyl sulphonyl fluoride (PMSF)]. Following the homogeniztion procedure, smples were incuted on ice for 2 min. Centrifugtion ws then crried out t 72 g t 4 for min. The nucler pellet ws dispersed y the uffer nd pssed through 21G needle 2 times. The smples were then centrifuged gin t 72 g t 4 for 1 min. After removing the superntnt, the nucler pellet ws re-suspended in μl nucler lysis uffer contining 2 mmol/l Tris (ph 7.), 137 mmol/l NCl, 2 mmol/l EDTA, 1 mmol/l CCl2, 1 mmol/l MgCl2, 1% Nonidet P-4 (NP-4), 2 mmol/l sodium orthovndte, 1 mmol/l sodium fluoride, 1 mmol/l sodium pyrophosphte, 1% glycerol,.1% sodium dodecyl sulfte (SDS) nd the forementioned protese inhiitors. The nucler pellet ws sonicted riefly for 3 s nd sved for western lotting. For mitochondril frctiontion whole cell lyste fter removing the nuclei ws centrifuged t 1 g, 4 for 1 min nd sved the pellet s mitochondril frction t -8. MDA nlysis in serum nd tissues MDA level ws determined y the thiorituric cid (TBA) method [22]. TBA rection ws performed ccording to mnufctory guidnce. To ssess MDA level in serum, 1 L serum ws used. For tissue MDA ssy out 3 mg of tissues (skeletl muscle, liver or dipose tissue) or mitochondri extrcted from 3 mg skeletl muscle were homogenized in 3 L ice-cold PBS. TBA rection ws performed ccording to mnufctory guidnce. Briefly, smples were incuted with TBA nd SDS t 9 for 1 h, followed y centrifugtion t 8 g for 1 min. Superntnts were trnsferred to 96-well plte nd the sornce ws mesured t 32 nm. The protein concentrtion in smples ws determined y BCA ssy kit. Serum MDA level ws clculted ccording to the formul provided y the compny guidnce, i.e., serum MDA (nmol/ml) = (smple OD vlue-ckground OD)/( Stndrd OD-lnk OD) stndrd concentrtion (1 nmol/ml) smple dilution times, while tissue MDA level fter the clcultion ws further corrected y smple protein concentrtion (mg protein/ml). MDA equivlents were expressed s nmol/mg tissue protein or nmol/ml serum. ROS mesurements in tissues Skeletl muscles were homogenized nd ROS level nlysis ws performed using 2,7 -dichlorofluorescein dicette fluorescent dye s proe nd fluorescence density ws mesured t 49/2 nm ccording to mnufctory guidnce nd the mesured vlues of opticl density [ reltive fluorescence unit (RFU)] were corrected y the protein concentrtions of smples nd were expressed s RFU/ g protein. Insulin nd glucose mesurements nd homeostsis model ssessment - insulin resistnce index After the mice were fsted for 6 h, lood smples were collected. To ssess plsm insulin levels, n ELISA sed method (Mercodi AB, Uppsl, Sweden) ws used, the method ws ccording to mnufctory guidnce nd expressed s g/l. Plsm glucose ssy levels were determined using glucose ssy kit from Sigm-Aldrich (Sint Louis, United Sttes), with the method provided y the mnufcturer. The level of the originl plsm glucose concentrtion ws mesured t 4 nm nd expressed s mg/dl. Homeostsis model ssessment- insulin resistnce index (HOMA-IR) ws clculted s [fsting plsm glucose (mmol/l) fsting serum insulin ( IU/mL)] /22. [23]. Tissue protein preprtion nd western lotting Methods for tissue protein preprtion nd western lotting were performed s descried [19]. A frction of liver, dipose tissue nd skeletl muscle were homogenized in ice-cold lysis uffer contining 2 mmol/l Tris (ph 7.), 137 mmol/l NCl, 2 mmol/l EDTA, 1 mmol/l CCl2, 1 mmol/l MgCl2, 1% NP-4, 2 mmol/l sodium orthovndte, 1 mmol/l sodium fluoride, 1 mmol/l sodium pyrophosphte, 1% glycerol, 2 g/ml protinin, g/ml leupeptin nd 2 mmol/l PMSF. The homogentes were then sonicted on ice cold conditions three times for 2 s nd centrifuged t 12 rpm for 1 min t 4. The superntnt ws collected. The protein concentrtion ws determined y BCA ssy kit. Equl mounts of proteins were used for western lot nlysis. The proteins were heted t 9 for min in SDS smple loding uffer nd underwent SDS-PAGE nlysis s descried in the following. The cellulr proteins were seprted y SDS-PAGE (1%) nd trnsferred to nitrocellulose memrnes. Following the trnsfer, the memrnes were incuted for 1. h t room temperture in the uffer contining 2 mmol/l Tris (ph 7.6), 14 mmol/l NCl,.1% Tween-2 nd % skimmed milk. The memrnes were then proed with ntiody overnight t 4. The memrnes were wshed nd incuted with secondry ntiody conjugted to horserdish peroxidse. Immunorective proteins were visulized with ECL. The intensities of the nds were quntified y phosphorimger nlysis using NIH imge softwre. 96 My 1, 212 Volume 3 Issue

4 Body weights (g) He HJ et l. Curcumin ctivtes Nrf2 nd ttenutes insulin resistnce A curcumin c B t/wk Adipose weight (g) C Liver weight (g) Sttisticl nlysis All the vlues were expressed s men ± SE. Sttisticl comprisons of mens mong three groups were crried out y using nlysis of vrince nd the P vlue less thn. ws considered to e sttisticlly significnt. d + curcumin + curcumin Figure 1 Effects of short term curcumin gvge on weights of whole ody, epididyml ft tissue nd liver. A: Body weight. The ody weight of mice ws mesured weekly following norml diet or high ft diet feeding during the period of 18 wk. The rrow indictes the strting time of curcumin gvge; B: Weight of epididyml dipose tissue; C: Liver weight. The mice were fed with either norml diet (, n = 1) or high ft diet (, n = 2) for 16 wk. The fed mice then received gvge of either curcumin ( mg/kg per dy, n = 1) or vehicle (1% croxymethyl cellulose uffer, n = 1) for 1 d. The fed mice were lso gvged with the vehicle. The mice were scrificed for tissue isoltion nd weighing. P <.1, or + curcumin vs ; P <.1, + curcumin vs ; c P <.1 + curcumin vs ; d P <., + curcumin vs. Four mice from nd four mice from + curcumin group died during the gvge procedure. The correltion etween re under the curve (AUC) nd MDA or ROS ws tested with the method of multiple liner regressions. RESULTS Short-term curcumin tretment does not lter BW Firstly, we fed the mice with to induce oesity nd insulin resistnce. As shown in Figure 1A, mice who received feeding exhiited more rigorous BW gin when compred with the mice of the group. The difference reched sttisticl significnce t 11 wk fter feeding. Curcumin ws supplemented fter 16 wk feeding during consumption, which ws different from the previous studies when curcumin ws dministered t the eginning of feeding nd prevented -induced oesity [17,18]. A short-term nd low dose of curcumin dministrtion ws designed to void its BW lowering effect in order to detect the primry mechnism of its ction. We gvged group of -fed mice with mg/kg BW of curcumin consecutively for 1 d. The control mice received the vehicle solution. This curcumin tretment did not lter BW gin in mice while fed with (Figure 1A). To further detect its effect on oesity, the weight of epididyml ft pds (n indictor of viscerl ft mss) ws mesured. We oserved tht induced out n 8-fold increse in the weight of epididyml ft pds compred to mice (:.21 ±.7 g vs : 1.6 ±.12 g, P <.1, Figure 1B), while curcumin gvge only modertely reduced the weight of epididyml ft in mice who received ( + curcumin: 1.19 ±.2 g vs : 1.6 ±.12 g, P <., Figure 1B) nd compred with mice in the group, the increse of the weight of epididyml ft in curcumintreted mice ws still out -fold (:.21 ±.7 g vs + curcumin: 1.19 ±.2 g, P <.1, Figure 1B). We lso mesured the weight of the liver nd there ws no sttisticl difference mong the three groups (Figure 1C). Curcumin improves glucose disposl nd insulin sensitivity As indicted previously, curcumin dministrtion signifi- 97 My 1, 212 Volume 3 Issue

5 AUC (mmol/l 12 min) Serum insulin ( IU/mL) He HJ et l. Curcumin ctivtes Nrf2 nd ttenutes insulin resistnce A 3 + curcumin Blood glucose (mmol/l) e t/min B 3 c C d e f 1 2 D + curcumin E + curcumin Serum glucose (mmol/l) curcumin HOMA-IR e + curcumin Figure 2 Effects of short term curcumin gvge on glucose tolernce nd insulin sensitivity. A: Intrperitonel glucose tolernce test in the norml diet group (n = 1), high ft diet group (n = 6) nd high ft diet plus curcumin group (n = 6); B: Are under the curve of glucose tolernce test; C: Are under the curve; D: Fsting plsm glucose levels; E: Homeostsis model ssessment - insulin resistnce index vlues. The mice were fed with different diets nd gvged with curcumin s indicted in Figure 1. At the end of 18 wk following norml diet (), high ft diet () nd +curcumin tretments, intrperitonel glucose tolernce test ws performed nd lood glucose levels were mesured. P <.1, or + curcumin vs t ech time point; P <.1, vs ; c P <.1, vs ; d P <., + curcumin vs ; e P <.1, + curcumin vs ; f P <., +curcumin vs. AUC: Are under the curve; HOMA-IR: Homeostsis model ssessment- insulin resistnce index. cntly prevented -induced insulin resistnce y longterm supplementtion [17]. To ssess whether curcumin could reverse insulin insensitivity in n estlished oese model or to test its tretment efficcy, we performed n intrperitonel glucose tolernce test (IPGTT). As shown in Figure 2A, fter 18 wk feeding of, glucose levels following the i.p. injection were significntly higher when compred with those in -fed mice. Curcumin tret- 98 My 1, 212 Volume 3 Issue

6 He HJ et l. Curcumin ctivtes Nrf2 nd ttenutes insulin resistnce A 2 B 4 Serum MDA (mmol/l) Muscle MDA (mmol/l) C 6 + curcumin D + curcumin Muscle ROS (RFU/ g) Mitochondril MDA (mmol/mg) curcumin + curcumin Figure 3 Effects of short term gvge of curcumin on mlondildehyde levels. A: Serum mlondildehyde levels; B: Mlondildehyde levels in skeletl muscle; C: Rective oxygen species level in whole cell lystes of skeletl muscle; D: Mlondildehyde levels in mitochondri of skeletl muscle. The mice were fed with indicted diets nd gvged with curcumin or vehicle. At the end of 18 wk following norml diet (, n = 1), high ft diet (, n = 6) nd + curcumin (n = 6) tretments, the mice were scrificed for lood nd tissue collections. Serum ws isolted, tissues were homogenized nd mitochondri were isolted. Mlondildehyde ssessments or rective oxygen species ssy were performed s descried in Mterils nd Methods. P <.1, vs ; P <.1 + curcumin vs ; c P <.1, + curcumin vs. MDA: Mlondildehyde; ROS: Rective oxygen species. ment reduced lood glucose levels (Figure 2A). The nlysis of AUC of the IPGTT during the tested 12 min lso showed tht induced out 2-fold increse in the AUC index compred to tht in mice, while curcumin normlized this elevtion y more thn % (Figure 2B). Furthermore, in order to ssess whether insulin sensitivity ws enhnced y curcumin, we mesured fsting glucose nd insulin levels (Figure 2C nd D) nd clculted the HOMA-IR index (Figure 2E). We found tht induced lmost 3-fold increse in this index while curcumin significntly reversed this normlity. These results indicte tht the effect of curcumin on improving glucose disposl is vi enhncing insulin ction nd this ction of curcumin in -fed mice is disposle with its effect on ttenuting BW gin. Curcumin meliortes -induced oxidtive stress in skeletl muscle nd mitochondri Incresed evidence indictes tht oxidtive stress plys cusl role in the development of insulin resistnce [7,24,2]. We tested whether nd curcumin lter redox lnce y ssessing MDA levels, stle indictor of oxidtive stress [22]. As shown in Figure 3A, induced pproximtely 2% elevtion of serum MDA level, while short-term curcumin gvge completely reversed this elevtion (: ±. nmol/ml vs + curcumin: ±.91 nmol/ml, P <.1). We then ssessed MDA levels in the insulin sensitive tissues. MDA level in the skeletl muscle of -fed mice ws incresed pproximtely 2-fold when compred with tht in the mice (Figure 3B). Curcumin dministrtion decresed musculr MDA content to the level tht ws comprle with tht in the mice (Figure 3B). Furthermore, musculr ROS content ws incresed out 4% y feeding while curcumin tretment completely locked this elevtion (: ± 4.3 RFU/ g vs : 1.7 ± 4.92 RFU/ g vs P <.1; : 1.7 ± 4.92 RFU/ g vs + curcumin: 3.18 ± 6.66 RFU/ g, P <.1, Figure 3C). Since mitochondril oxidtive stress plys mjor custive role in insulin resistnce in the condition of nutrition over consumption [4,8], we mesured the MDA level in the mitochondril frction of skeletl muscles. 99 My 1, 212 Volume 3 Issue

7 He HJ et l. Curcumin ctivtes Nrf2 nd ttenutes insulin resistnce A 7 B curcumin MDA of dipose tissue (nmol/mg) Liver MDA (nmol/mg) + curcumin Figure 4 Effects of curcumin on dipose nd liver mlondildehyde levels. A: Mlondildehyde levels in dipose tissue; B: Mlondildehyde levels in liver. The mice were fed with indicted diets nd gvged with curcumin or vehicle. At the end of 18 wk following norml diet (, n = 1), high ft diet (, n = 6) nd + curcumin (n = 6) tretments, the mice were scrificed, tissues were homogenized nd mlondildehyde levels were mesured s indicted in the Mterils nd Methods. P <.1, or +curcumin vs. MDA: Mlondildehyde. Indeed, the MDA level in the mitochondril frction ws pronouncedly elevted in the -fed mice (4-fold elevtion compred to mice) nd curcumin significntly ttenuted the effect of (: 33.7 ± 1.16 nmol/mg vs + curcumin: 1. ± 2.9 nmol/ mg, P <.1, Figure 3D). We, however, did not detect chnge of MDA level in liver or dipose tissue y curcumin gvge (Figure 4). By performing regression nlysis on the musculr MDA level nd the AUC vlue in IPGTT, we found tht there ws significntly positive correltion etween them (P <.). The regression nlysis ws lso performed on the musculr mitochondril MDA level nd the AUC vlue in IPGTT. A more significntly positive correltion ws oserved (P <.1). These dt suggest the existence of tight link etween musculr mitochondril redox lnce nd the ility of the whole ody on glucose disposl. Curcumin ctivtes Nrf2 signling in skeletl muscles Curcumin hs een shown to ctivte the Nrf2 system in cell culturing systems [1]. To further explore the potentil mechnism of n nti-oxidtive effect of curcumin, we determined whether curcumin stimultes Nrf2 signling in vivo. We prepred whole lystes of tissues from the three groups of mice nd exmined the levels of Nrf2 s well s one of its trget gene products, HO-1 y western lotting. As shown in Figure A, when compred with the control mice, feeding reduced Nrf2 level pproximtely % in muscles, wheres curcumin dministrtion significntly ttenuted the repressive effect of (Figure A). We however, did not see n pprent chnge in the protein levels of Nrf2 nd HO-1 in the liver nd the ft tissue y this short-term curcumin tretment (Figure C nd D). Consistently, Nrf2 content in the nuclei of skeletl muscle ws reduced in mice while curcumin tretment reversed this reduction (Figure B). Furthermore, feeding led to reduced level of HO-1 wheres curcumin significntly reversed this reduction. These results collectively suggest tht feeding impirs the function of Nrf2 system while short-term tretment with curcumin significntly ctivtes the Nrf2-ARE signling pthwy in skeletl muscles of -fed mice. DISCUSSION Long-term curcumin dministrtion in mice hs een shown to improve insulin signling nd glucose disposl y ttenuting inflmmtion nd oesity [17,26]. However, it is not cler whether curcumin could exert its insulinsensitizing ction y nti-oxidtive stress. Prticulrly, very few studies hve een conducted to exmine the cute effect of curcumin in n lredy insulin resistnt model. We showed here tht short-term curcumin gvge improved glucose disposl in -induced mouse model in the sence of n pprent chnge of BW. We then oserved tht short-term curcumin dministrtion meliorted oxidtive stress in oth serum nd muscles of mice, prticulrly in musculr mitochondri. Furthermore, we detected tht these eneficil effects were ccompnied y reversl of impired Nrf2 signling, including incresed Nrf2 expression, its nucler loction nd the expression of the trget ntioxidnt enzyme, HO-1. We hence suggest tht the ctivtion of the mjor nti-oxidtive defense mchinery Nrf2 in muscle is novel mechnism for curcumin in the tretment of insulin resistnce nd ssocited metolic disorders. In -induced insulin resistnt model, severl mechnisms hve een proposed to explin the cusl role of consumption in the development of insulin insensitivity. Inflmmtion, ER stress nd mitochondril dysfunction re ll reported to e involved [3,6-8]. However, these normlities could e the chronic etiology fctors of insulin resistnce. It is still not known which fctor initilly induces insulin resistnce. It is resonle to elieve tht in the condition of nutrition over supply, mitochondril oxidtive phosphoryltion metolism would produce lrger mount ROS nd if nti-oxidtive mchinery could not eliminte the mitochondril derived oxidnts, oxidtive stress will occur, resulting in insulin 1 My 1, 212 Volume 3 Issue

8 Nrf2 (opticl density) Nrf2 (opticl density) Nrf2 (opticl density) He HJ et l. Curcumin ctivtes Nrf2 nd ttenutes insulin resistnce A Muscle + curcumin C Liver + curcumin Totl Nrf2 Nrf2 HO-1 HO-1 -ctin -ctin 1..8 c d HO-1 (opticl density) curcumin d HO-1 (opticl density) curcumin + curcumin + curcumin D Adipose tissue + curcumin B Nrf2 Muscle nuclei + curcumin Nrf2 HO-1 Hostine H3 -ctin Nucler Nrf2 (opticl density) d + curcumin c + curcumin Figure Effects of curcumin on nucler fctor erythroid-2-relted fctor-2 signling. A: The protein levels of nucler fctor erythroid-2-relted fctor-2 (Nrf2), heme oxygense-1 (HO-1) in skeletl muscle (β-ctin s the loding control of totl protein); B: Nucler Nrf2 contents in skeletl muscle were proed with Nrf2 ntiody (Histone H3 s the nucler protein loding control). C, D: The levels of Nrf2 nd HO-1 in livers were exmined y western lotting. The nlysis of reltive opticl density on western lots ws presented in the lower pnels or the right pnel for corresponding figures. The mice were fed with indicted diets nd gvged with curcumin or vehicle. At the end of 18 wk, the mice were scrificed nd tissues were collected for protein or nucler preprtion. Western lotting ws performed with indicted ntiodies. P <., P <.1, c P <.1, or +curcumin vs ; d P <., + curcumin vs. : High ft diet; : norml diet. HO-1 (opticl density) curcumin 11 My 1, 212 Volume 3 Issue

9 He HJ et l. Curcumin ctivtes Nrf2 nd ttenutes insulin resistnce resistnce. In support, recent investigtions hve indicted tht muscle mitochondril derived ROS cn cutely impir glucose disposl [27,28] nd the dministrtion of mitochondril specific ntioxidnt corrects this defect [7,2]. Interestingly, curcumin hs n nti-oxidnt effect nd in vitro studies hve indicted its ction on triggering Nrf2 signling [14,1]. We therefore proposed the working hypothesis of this study tht short-term curcumin supplementtion could improve glucose homeostsis in mice y its nti-oxidtive ction vi Nrf2 ctivtion. Firstly, we exmined the redox sttus in the three groups of mice y MDA ssy. We demonstrte here tht induces severe oxidtive stress in skeletl muscle especilly in mitochondri, while curcumin dministrtion reverses this deleterious effect. The regression nlysis on the AUC dt of IPGTT nd MDA level hs further scertined the reltionship of muscle mitochondril redox sttus with insulin sensitivity. We hence suggest tht musculr mitochondri re initil nd importnt working sites for curcumin in ttenuting oxidtive stress nd improving glucose tolernce. Detiled mechnisms underlying mitochondril redox sttus in regulting insulin ction re currently uncler. One possiility is tht ROS impirs mitochondril function, followed y reduced lipid oxidtion nd incresed lipid ccumultion in muscle. The elevtion of muscle lipid content then ctivtes protein kinse C, which locks insulin receptor sustrte-1 phosphoryltion nd downstrem insulin signling [29]. Further experiments re needed to ssess whether curcumin is le to improve insulin signling vi locking this pthologicl signling pthwy. Redox lnce is determined y oth ROS production nd nti-oxidtive function (ROS elimintion). Recent studies hve indicted tht n inility of the Nrf2 system in the elimintion of ROS leds to the development of insulin resistnce, wheres phrmcologicl or genetic ctivtion of Nrf2 cn improve insulin signling, long with improved glucose metolism [19,3]. To further investigte the underlying mechnism of curcumin on regulting redox lnce, we exmined Nrf2 signling in insulin sensitive tissues. We found tht repressed Nrf2 function in muscles y reducing oth totl Nrf2 content nd its nucler portion, wheres curcumin mrkedly reversed these defects. Furthermore, curcumin lso incresed the expression of HO-1, downstrem trget of Nrf2 nd n essentil nti-oxidtive enzyme. These dt re consistent with the reversl of oxidtive stress in muscle ut not in ft tissue or the liver. Therefore, these results suggest tht curcumin-induced Nrf2 ction is novel mechnism ginst -induced oxidtive stress nd to ttenute glucose intolernce. In line with the positive regultion of the Nrf2/HO-1 system on insulin sensitivity, HO-1 hs een shown to repress oxidtive stress nd inflmmtion [31], nd the dministrtion of HO-1 inducer improves insulin sensitivity [32,33]. Due to the tight link of oxidtive stress with inflmmtion [31], further studies should e conducted to ddress whether the ctivtion of Nrf2 y curcumin is relted to its nti- inflmmtory effect. It should lso e pointed out tht lthough 1 d curcumin supplementtion did not reduce the BW in -fed nimls, we egn to see the effect of curcumin on reducing ft mss. We hence cnnot rule out the possiility tht curcumin-evoked Nrf2 signling modulte lipid metolism, dipogenesis, dipocyte differentition, indirectly ffecting glucose metolism [17-19]. Curry contining curcumin, populr nturl spice in Indi, hs een used s trditionl drug to tret inflmmtion, wheres modern studies further demonstrte the potentil of curcumin to tret other mjor lifethretening diseses, such s hert filure, neurodegenertive diseses nd dietes [13,17,18,34]. The reltive high sfety nd low cost of curcumin would lso encourge its therpeutic pplictions in the future. In ddition to the preventive effect of curcumin on insulin resistnce, we demonstrted here tht fter the induction of oesity y, short-term curcumin gvge still reversed glucose intolernce, clerly suggesting the therpeutic ppliction of curcumin in the tretment of insulin resistnce-relted metolic disorders. In light of its clinicl usge, studies hve een conducted nd verified the effectiveness of curcumin in treting hyperglycemi-induced oxidtive stress nd relted disorders, such s dietic nephropthy [3,36]. In ccordnce, the present study provides significnt mechnism of Nrf2 ctivtion y which curcumin cn defend oxidtive stress nd mitochondril redox imlnce nd ttenute the normlity of glucose metolism in the condition of nutritionl oversupply. Together these findings would plce Nrf2 nd curcumin s the new therpeutic trgets nd pproches for the tretment of hyperglycemi, s well s oxidtive stressrelted diseses. COMMENTS Bckground Curcumin intervention ttenutes insulin resistnce nd improves glucose dis- posl invriousrodentmodelsofinsulinresistncenddietes.thecurrent rodent of insulin resistnce nd dietes. current mechnistic understnding on these eneficileffectsislimitedlthough effects islimitedlthough curcumin hseenshowntottenuteodyweight(bw) shown ttenute ody weight ginndinflmmtorynd inflmmtory response during high ft diet consumption. Whether curcumin improves insulin signling vi ctivting the endogenous nucler fctor erythroid-2-relted fctor-2 (Nrf2) nti-oxidtive stress system nd whether the improvement involves the ttenution of mitochondril oxidtive stressreunknown. Reserch frontiers The use of nturlly occurring compound in intervention is n optimum pproch for the tretment nd prevention of dietes nd oesity. Mitochondri re primry site of production of free rdicls. Extensive recent studies hve reveled the fundmentl importnce of mitochondril oxidtive stress in the etiology of insulin resistnce. The ctivtion of the mjor nti-oxidtive stress systemnrf2wsshowntoimproveinsulinsignlingndglucosedisposl shown to insulin signling nd glucose disposl in vivo. Curcumin is le to ctivte Nrf2 signling in vitro. Here the uthors ddressed the question whether curcumin, nturlly-occurring compound, improves insulin signling vi ttenuting oxidtive stress in mitochondri, vi ctivting the Nrf2 signling pthwy. Innovtions nd rekthroughs The study showed hereforthefirsttimethestimultoryeffectofcurcuminincti- first time stimultory effect of curcumin in ctivting endogenous Nrf2 system in vivo. This finding will initite further investigtions on mechnistic explortion of the stimultion of Nrf2 system y nturllyoccurring plnt compounds. 12 My 1, 212 Volume 3 Issue

10 He HJ et l. Curcumin ctivtes Nrf2 nd ttenutes insulin resistnce The uthors demonstrtedherethtcurcuminimprovedinsulinsignling tht improved insulin signling nd glucose disposl, ssocited with the ttenution of muscle mitochondril oxidtive stress. This finding defined muscle mitochondri s novel trgets for curcumin. The improvement oserved in this niml model is independent of the BW lowering effect of curcumin. The uthors hve hence not only deepened the mechnistic understnding of the therpeutic effect of curcumin, ut lso reveled tht ttenution of endogenous nti-oxidtive stress is n importnt pthologicl event of high ft diet consumption. Applictions This studyidentifiednewtrgetforcurcuminndothernturlly-occurring new trget for nd other nturlly-occurring compounds in improving insulinsensitivity.itfurthersupportsthehypothesistht sensitivity. supports the hypothesis tht curcumin cn e utilized in the tretment nd prevention of dietes nd other metolic disorders tht involve insulin resistnce. Terminology Insulin resistnce is the pthophysiologicl condition tht insulin cn not exert norml function on itstrgetingtissuesorcells,includingmuscle,livernd trgeting tissues including,livernd dipose tissue, or higher concentrtion of insulin is required to evokeinsulin signling nd glucose uptke. The normlityisthesisforthedevelopment of insulin-resistnt diseses, including type2dietesmellitus,hypertension nd certin crdiovsculr diseses.nrf2 nti-oxidtive system:in thecondition of oxidtive stress, cytosol Nrf2 will e trnslocted into the nucleus.it Itwillthen then ind to ttery of specifictrgetgenesintheirpromoterregions,stimultingthe genes in theirpromoterregions,stimultingthe regions, stimulting trnscription of these genes. Nrf2 trget genes encode series of nti-oxidnt enzymes, including HO-1. Peer review The present study showed thtshorttermtretmentwithcurcuminctivtesnrf2 term tretment ctivtes signling nd lowers lood glucose levels nd oxidtive stress in C7BL/6J mice fed on high-ft diet. It hs two strengths. Firstly,,demonstrtionofNrf2ctivtion of ctivtion nd nucler trnsloction following curcumin tretment in n niml model of high-ft diet induced oesity nd insulin resistnce. Secondly,,demonstrtionof of reversl of high ft diet-induced mlondildehyde elevtions in serum, muscle nd muscle mitochondri. REFERENCES 1 Reven GM. Insulin resistnce: the link etween oesity nd crdiovsculr disese. Med Clin North Am 211; 9: Asi F, Brown BW, Lmendol C, McLughlin T, Reven GM. Reltionship etween oesity, insulin resistnce, nd coronry hert disese risk. J Am Coll Crdiol 22; 4: Wellen KE, Hotmisligil GS. In flmmtion, stress, nd dietes. J Clin Invest 2; 11: Mrtínez JA. Mitochondril oxidtive stress nd in flmmtion: n sllom to oesity nd insulin resistnce. J Physiol Biochem 26; 62: Light DW, Desi KM, Gopul NK, Anggård EE, Crrier MJ. Pro-oxidnt chllenge in vivo provokes the onset of NIDDM in the insulin resistnt oese Zucker rt. Br J Phrmcol 1999; 128: Rins JL, Jin SK. 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11 He HJ et l. Curcumin ctivtes Nrf2 nd ttenutes insulin resistnce 29 Roden M. Muscle triglycerides nd mitochondril function: possile mechnisms for the development of type 2 dietes. Int J Oes (Lond) 2; 29 Suppl 2: S111-S11 3 Tn Y, Ichikw T, Li J, Si Q, Yng H, Chen X, Goldltt CS, Meyer CJ, Li X, Ci L, Cui T. Dietic downregultion of Nrf2 ctivity vi ERK contriutes to oxidtive stress-induced insulin resistnce in crdic cells in vitro nd in vivo. Dietes 211; 6: Pine A, Eiz-Vesper B, Blsczyk R, Immenschuh S. Signling to heme oxygense-1 nd its nti-in flmmtory therpeutic potentil. Biochem Phrmcol 21; 8: Li M, Kim DH, Tsenovoy PL, Peterson SJ, Rezzni R, Rodell LF, Aronow WS, Ikehr S, Arhm NG. Tretment of oese dietic mice with heme oxygense inducer reduces viscerl nd sucutneous diposity, increses diponectin levels, nd improves insulin sensitivity nd glucose tolernce. Dietes 28; 7: Nicoli A, Li M, Kim DH, Peterson SJ, Vnell L, Positno V, Gstldelli A, Rezzni R, Rodell LF, Drummond G, Kusmic C, L Ate A, Kpps A, Arhm NG. Heme oxygense-1 induction remodels dipose tissue nd improves insulin sensitivity in oesity-induced dietic rts. Hypertension 29; 3: Begum AN, Jones MR, Lim GP, Morihr T, Kim P, Heth DD, Rock CL, Pruitt MA, Yng F, Hudspeth B, Hu S, Full KF, Teter B, Cole GM, Frutschy SA. Curcumin structurefunction, iovilility, nd ef ficcy in models of neuroinflmmtion nd Alzheimer s disese. J Phrmcol Exp Ther 28; 326: Khjehdehi P, Pkfetrt M, Jvidni K, Azd F, Mlekmkn L, Ns MH, Dehghnzdeh G. Orl supplementtion of turmeric ttenutes proteinuri, trnsforming growth fctor-β nd interleukin-8 levels in ptients with overt type 2 dietic nephropthy: rndomized, doule-lind nd plceo-controlled study. Scnd J Urol Nephrol 211; 4: Rem M, Prdeep R. Dietic retinopthy: n Indin perspective. Indin J Med Res 27; 12: S- Editor Wu X L- Editor Roemmele A E- Editor Wu X 14 My 1, 212 Volume 3 Issue