JPFSM: Short Communication. Abstract Maturation and aging induce alterations in glucose and protein metabolism, which

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1 J Phys Fitness Sports Med, 2(4): (2013) DOI: /jpfsm JPFSM: Short Communiction Effects of long-term supplementtion with tetrhydrocurcumin nd rnched-chin mino cids on glucose tolernce nd muscle protein content in mture rts Mik Mochizuki 1, Hiroshi Tkyngi 2, Snshiro Ymd 2, Toshihiko Osw 1, Ysuyuki Kitur 2 nd Yoshihru Shimomur 2* 1 Deprtment of Helth nd Nutrition, Fculty of Psychologicl nd Physicl Science, Aichi Gkuin University, 12 Arike, Iwski-cho, Nisshin, Aichi , Jpn 2 Lortory of Nutritionl Biochemistry, Deprtment of Applied Moleculr Biosciences, Grdute School of Biogriculturl Sciences, Ngoy University, Furo-cho, Chikus-ku, Ngoy, Aichi , Jpn Received: August 1, 2013 / Accepted: August 20, 2013 Astrct Mturtion nd ging induce ltertions in glucose nd protein metolism, which re responsile for insulin resistnce nd srcopeni. In the present study, we exmined the effects of long-term (16 weeks) ingestion of diets supplemented with tetrhydrocurcumin (THC) nd/or rnched-chin mino cids (BCAAs) on glucose tolernce nd soleus muscle protein content in mture rts (28 weeks of ge). Intrperitonel glucose tolernce tests (IPGTTs) were performed t week 6 nd week 12 during the experimentl period. Glucose tolernce ws not ffected y 6-week supplementtion with THC nd/or BCAAs, ut ws improved y supplementtion t 12 weeks. A synergistic effect of THC nd BCAAs ws not oserved. The protein content of the soleus muscle ws incresed y long-term supplementtion with BCAAs, ut not THC. These results suggest tht THC nd BCAAs re potentilly eneficil supplements to improving mturtion (ging)-relted metolic deteriortion. Keywords : tetrhydrocurcumin, rnched-chin mino cids, glucose tolernce, soleus muscle protein, mture rts Introduction The complex physiologicl process of ging 1) is chrcterized y incresed production of rective oxygen species reltive to the ntioxidnt defenses in niml cells 2,3). It hs een reported tht dministrtion of ntioxidnts, such s curcumin, to ging nimls is effective for decresing ging-ssocited phenotypic chnges 4,5). Tetrhydrocurcumin (THC), one of the mjor metolites of curcumin, is structurlly similr to curcumin in tht they hve identicl diketone structures nd phenolic groups, ut differs in tht it lcks the doule onds of curcumin 6). Recent ttention hs een focused on THC, ecuse this compound ppers to exert greter ntioxidnt ctivity thn curcumin in oth in vitro nd in vivo systems 6,7). Sugiym et l. reported tht THC exhiited similr physiologicl nd phrmcologicl properties s the ctive form of curcumin in vivo 8). One of the phenotypic chnges ssocited with ging is muscle loss (srcopeni), which results from n imlnce etween protein synthesis nd degrdtion rtes 3). It hs een reported tht ged muscle is less sensitive to stimultion of protein synthesis y physiologicl concentrtions *Correspondence: shimo@gr.ngoy-u.c.jp of insulin nd mino cids, ut is still le to respond to superphysiologicl concentrtions of insulin nd mino cids 9-12). It hs een demonstrted tht rnched-chin mino cids (BCAAs, especilly leucine) ply n importnt role in regultion of protein metolism y stimulting protein synthesis through ctivtion of mmmlin trget of rpmycin (mtor) complex 1, nd suppressing protein degrdtion y inhiiting utophgy 13). Furthermore, ccumulting evidence indictes tht BCAAs hve importnt roles in glucose metolism, especilly in glucose tolernce 14-16). Bsed on the findings descried ove, it is possile tht tretment of ging nimls with THC nd BCAAs might e effective for decresing ging-relted phenotypic chnges. Furthermore, it is interesting to exmine the synergetic effects of these supplements, ecuse they my ct y different mechnisms. Therefore, we exmined the effects of the long-term ingestion of diets supplemented with THC nd/or BCAAs on glucose tolernce nd muscle protein content in mture rts. Mterils nd Methods All procedures were pproved y the Animl Cre Committee of Ngoy University Grdute School of

2 510 JPFSM: Mochizuki M, et l. Biogriculturl Sciences. Twenty-four mle Wistr rts ged ~24 weeks were purchsed from Jpn SLC (Hmmtsu, Jpn). The nimls were individully housed in wire-mesh cges in conventionl niml room with controlled temperture (22 ± 1 C) nd 12-h light/drk cycle (light from 08:00 ech dy). Four experimentl diets (Tle 1) were prepred in pellet form y CLEA Jpn: control, THC, BCAA, nd THC+BCAA comined diet. The composition of ech experimentl diet ws sed on the AIN-93M diet 17) ; nd THC (0.2%, Nikken Fine Chemicls, Shizuok, Jpn) nd the mino cid mixture (5% BCAAs or non-essentil mino cids, Ajinomoto Co, Inc, Tokyo, Jpn) supplements were replced with the sme mount of cornstrch in the AIN-93M diet (Tle 1). The rtio of BCAAs (leucine : isoleucine: vline) ws 2 : 1 : ). In order to djust the nitrogen content in ll of the experimentl diets, mixture of non-essentil mino cids sed on the mino cid composition of csein 17) ws dded to the control nd THC diets. Rts were rndomly divided into four diet groups (n=6 ech): Control, THC, BCAA, nd THC+BCAA group. Rts in ech diet group were fed the corresponding experimentl diets for 16 weeks. Food intke nd ody weight (BW) were recorded twice week. During the experimentl period, intrperitonel glucose tolernce tests (IPGTT) were performed t week 6 nd week 12, s reported previously 16). On the finl dy of the experiment, rts were strved for ~8 h from 08:00, nesthetized y intrperitonel injection of sodium pentoritl (50 mg/kg BW), nd then scrificed y exsnguintion. The hilterl hindlim muscles (soleus, nd gstrocnemius+plntris muscles) were removed, freeze-clmped in liquid nitrogen, nd stored t -80 C until nlyses. The hert nd liver were lso otined nd weighed. The plsm glucose concentrtion ws ssyed enzymticlly using Glucose C-II kit, purchsed from Wko Pure Chemicl Industries, Ltd (Osk, Jpn). The soleus muscle is the predominnt slow-twitch oxidtive muscle nd is most intensively studied ecuse it is most ffected y disuse 19). We therefore mesured the solule nd myofirillr protein content in this muscle. Muscle protein frctiontion ws performed s descried previously 18), sed on the method of Grm et l. 20). The totl protein in the muscle ws the sum of solule nd myofirillr proteins. Protein content ws determined y the Brdford method 21) using ovine γ-gloulin s stndrd. Dt re presented s mens ± SE. Dt were nlyzed y ANOVA, followed y the Fisher s protected lest significnt difference test when the ANOVA ws significnt. P<0.05 ws considered to e sttisticlly significnt. Stt- View 5.0 softwre (SAS Institute Inc, Cry, NC) ws used for sttisticl nlyses of the dt. Results nd Discussion Body weight t the eginning, week 6, week 12, nd week 16 (the lst week) of the experimentl period did not differ mong ll of the diet groups, nd the weight of the soleus nd gstrocnemius+plntr muscles, hert, nd liver t the end of the experiment did not differ s well Tle 1. Composition of Control nd experimentl diets Ingredient Control diet BCAA THC THC+BCAA Corn strch Milk csein Dextrin Grnulted sugr Soyen oil Crystl cellulose Minerl AIN-93N Vitmin AIN-93N supplemented supplemented L-cysteine Choline itrtrte Non-essentil mino cid 5 5 BCAAs 5 5 THC Totl

3 JPFSM: Effects of THC nd BCAA on ging 511 mong the diet groups (dt not shown). Averge dily food intke during the experimentl period ws the sme mong ll of the diet groups (dt not shown), indicting tht supplementtion with THC nd/or BCAAs did not ffect food intke. IPGTTs were performed t week 6 (Fig. 1A) nd week 12 (Fig. 1B). The plsm glucose responses following glucose dministrtion did not differ etween the diet groups t week 6. During the IPGTT t week 12, however, the pek level of plsm glucose 30 min fter glucose dministrtion ws significntly higher in the control thn in the three other diet groups, ut ws not different mong the three experimentl diet (THC, BCAA, nd THC+BCAA) groups, suggesting tht glucose tolernce ws improved y supplementtion with THC nd/or BCAAs. When the glucose tolernce ws nlyzed using the re under the curve (AUC) of glucose concentrtions, the results showed similr trend s descried ove, ut were not significntly different mong the diet groups (AUC (mg/dl 2 h): 268 ± 20, 226 ± 16, 208 ± 37, nd 221 ± 19 for Control, THC, BCAA, nd THC+BCAA, respectively). The results otined in the present study re similr to those reported in studies in which streptozotocin-nicotinmide induced young dietic rts (8 weeks of ge) were orlly dministered dose of THC (80 mg/kg BW/ dy) for 45 dys 22), nd spontneous type 2 dietic rts (9 Control THC BCAA THC+BCAA Time fter ingestion (min) Fig. 1 Intrperitonel glucose tolernce test (IPGTT). IPGTTs were performed t week 6 (A) nd week 12 (B) during the experimentl period using the method descried in the text. *P<0.05 compred to mens of other groups t the sme time point. weeks of ge) were fed 5% BCAA diet for 7 weeks 23). The dose of THC or BCAAs used in these studies ws similr to tht used in the present study. Supplementtion of THC nd BCAAs for 6 weeks did not hve ny significnt effect on glucose tolernce in the present study, however, suggesting tht, compred to young rts, longerterm supplementtion my e required to see improvement in glucose tolernce in mture rts. A synergistic effect of THC nd BCAAs on glucose metolism ws not oserved in the present study, s glucose tolernce did not differ mong the three diet (THC, BCAA, THC+BCAA) groups. The effect of BCAAs on glucose tolernce my e ttriuted to the ctions of leucine nd isoleucine s reported previously 14,15). The solule protein content in the soleus muscle ws significntly higher in BCAA nd THC+BCAA groups thn in the Control diet group, ut ws not different etween the Control nd THC groups (Fig. 2A). A similr trend ws oserved with myofirillr protein content in muscle, ut the difference ws not significnt (Fig. 2B). The totl protein content in muscle ws significntly higher in the BCAA nd THC+BCAA diet groups thn in Control nd THC diet groups (Fig. 2C). These results suggest tht the protein content in the soleus muscle ws incresed y supplementtion with BCAAs, ut not THC. The protein content of the gstrocnemius+plntris muscles ws lso mesured in the present study, nd the results showed similr trend s those of the soleus muscle, lthough significnt difference ws not oserved mong the diet groups. Short-term hindlim suspension (for out 1 week) is well-estlished model for microgrvity, which induces pronounced trophy in nti-grvittionl muscles such s the soleus 19). We previously reported tht BCAA supplementtion during hindlim suspension tended to suppress decrese in protein content of the soleus muscle in the muscle trophy model of rts, ut did not ffect the decrese in soleus muscle weight 18). Although the supplementtion period ws much longer in the present study (16 weeks), we otined similr results in tht the protein content of the soleus muscle ws significntly higher in the BCAA diet group thn in the Control nd THC diet groups; ut muscle weight ws the sme mong the diet groups. In cses of long-term supplementtion, the mino cid content in the experimentl diet my e n importnt fctor ffecting protein metolism. Therefore, we djusted the mino cid content to e exctly the sme mong the experimentl diets. Since food consumption of rts did not differ mong the diet groups in the present study, the rts consumed the sme mount of dietry nitrogen. From these results, we cn conclude tht long-term supplementtion with BCAAs incresed the protein content in the soleus muscle of mture rts. It hs een shown tht rt plsm BCAA concentrtions re significntly elevted y the intke of BCAA-supplemented diet, ut not Control diet 18). Therefore, the effect of BCAA supplement-

4 512 JPFSM: Mochizuki M, et l. References, Fig. 2 Protein content of soleus muscle. Solule (A), myofirillr (B), nd totl (C) protein. The totl protein content ws clculted s sum of solule nd myofirillr proteins. Mens without common letter re significntly different (P<0.05). tion my e explined y the incresed intke of leucine tht stimultes protein synthesis nd inhiits protein degrdtion in muscle 13). Insulin sensitivity is suggested to e n importnt fctor ffecting muscle protein metolism, nd insulin resistnce develops during the ging process 13). In the present study, lthough BCAA supplementtion resulted in improved glucose tolernce nd incresed protein content of the soleus muscle, supplementtion with THC only improved glucose tolernce. These results imply tht the improvement of glucose tolernce oserved in this study might not contriute to the incresed protein content of the soleus muscle. Further studies re required to clrify the reltionship etween glucose tolernce nd muscle protein metolism during ging. Acknowledgments We thnk Nikken Fine Chemicls (Shizuok, Jpn) nd Ajinomoto Co, Inc, for providing THC (>99% pure) nd mino cids, respectively. 1) Kourtis N nd Tvernrkis N Cellulr stress response pthwys nd geing: intricte moleculr reltionships. EMBO J 30: ) Brzili N, Huffmn DM, Muzumdr RH nd Brtke A The criticl role of metolic pthwys in ging. Dietes 61: ) Dori E, Buonocore D, Focrelli A nd Mrztico F Reltionship etween humn ging muscle nd oxidtive system pthwy. Oxid Med Cell Longev 2012: ) Queen BL nd Tollefsol TO Polyphenols nd ging. Curr Aging Sci 3: ) Prk LK, Friso S nd Choi SW Nutritionl influences on epigenetics nd ge-relted disese. Proc Nutr Soc 71: ) Okd K, Wngpoengtrkul C, Tnk T, Toyokuni S, Uchid K nd Osw T Curcumin nd especilly tetrhydrocurcumin meliorte oxidtive stress-induced renl injury in mice. J Nutr 131: ) Pri L nd Murugn P Protective role of THC ginst erythromycin estolte induced heptotoxicity. Phrmcol Res 49: ) Sugiym Y, Kwkishi S nd Osw T Involvement of the diketone moiety in the ntioxidnt mechnism of tetrhydrocurcuminoids. Biochem Phrmcol 52: ) Drdevet D, Sornet C, Blge M nd Grizrd J Stimultion of in vitro rt muscle protein synthesis y leucine decreses with ge. J Nutr 130: ) Comret L, Drdevet D, Rieu I, Pouch MN, Béchet D, Tillndier D, Grizrd J nd Attix D A leucine-supplemented diet restores the defective postprndil inhiition of protesome-dependent proteolysis in ged rt skeletl muscle. J Physiol 569: ) Ktsnos CS, Koyshi H, Sheffield-Moore M, Arslnd A nd Wolfe RR A high proportion of leucine is required for optiml stimultion of the rte of muscle protein synthesis y essentil mino cids in the elderly. Am J Physiol Endocrinol Met 291: E381-E ) Fujit S, Glynn EL, Timmermn KL, Rsmussen BB nd Volpi E Suprphysiologicl hyperinsulinemi is necessry to stimulte skeletl muscle protein nolism in older dults: evidence of true ge-relted insulin resistnce of muscle protein metolism. Dietologi 52: ) Hnds SL, Proud CG nd Wyttench A mtor s role in geing: protein synthesis or utophgy? Aging (Alny NY) 1: ) Nishitni S, Tkehn K, Fujitni S nd Sonk I Brnched-chin mino cids improve glucose metolism in rts with liver cirrhosis. Am J Physiol Gstrointest Liver Physiol 288: G1292-G ) Doi M, Ymok I, Nkym M, Sughr K nd Yoshizw F Hypoglycemic effect of isoleucine involves incresed muscle glucose uptke nd whole ody glucose oxidtion nd decresed heptic gluconeogenesis. Am J Physiol Endocrinol Met 292: E1683-E ) Kdot Y, Kzm S, Bjotto G, Kitur Y nd Shimomur Y Clofirte-induced reduction of plsm rnchedchin mino cid concentrtions impirs glucose tolernce in rts. JPEN J Prenter Enterl Nutr 36: ) Reeves PG, Nielsen FH nd Fhey GC Jr AIN-93 puri-

5 JPFSM: Effects of THC nd BCAA on ging 513 fied diets for lortory rodents: finl report of the Americn Institute of Nutrition d hoc writing committee on the reformultion of the AIN-76A rodent diet. J Nutr 123: ) Bjotto G, Sto Y, Kitur Y nd Shimomur Y Effect of rnched-chin mino cid supplementtion during unloding on regultory components of protein synthesis in trophied soleus muscles. Eur J Appl Physiol 111: ) Bjotto G nd Shimomur Y Determinnts of disuseinduced skeletl muscle trophy: exercise nd nutrition countermesures to prevent protein loss. J Nutr Sci Vitminol 52: ) Grm T, Koyshi C, Hddd F, Adms GR, Bodell PW nd Bldwin KM Similr cute moleculr responses to equivlent volumes of isometric, lengthening, or shortening mode resistnce exercise. J Appl Physiol 102: ) Brdford MM A rpid nd sensitive method for the quntifiction of microgrm quntities of protein utilizing the principle of protein-dye inding. Anl Biochem 72: ) Murugn P nd Pri L Antioxidnt effect of tetrhydrocurcumin in streptozotocin-nicotinmide induced dietic rts. Life Sci 79: ) Kuzuy T, Ktno Y, Nkno I, Hirook Y, Itoh A, Ishigmi M, Hyshi K, Hond T, Goto H, Fujit Y, Shikno R, Murmtsu Y, Bjotto G, Tmur T, Tmur N nd Shimomur Y Regultion of rnched-chin mino cid ctolism in rt models for spontneous type 2 dietes mellitus. Biochem Biophys Res Commun 373: