(2013), 110, 981 987 q The Authors 2013 doi:10.1017/s0007114512006174 Post-exerise whey protein hydrolyste supplementtion indues greter inrese in musle protein synthesis thn its onstituent mino id ontent Atsushi Knd 1 *, Kyosuke Nkym 1, Tomoyuki Fuksw 1, Jinihiro Kog 1, Minoru Knege 1, Kentro Kwnk 2 nd Mitsuru Higuhi 3 1 Food Siene Reserh Lortories, Meiji Compny Limited, 540 Nrud, Odwr, Kngw 250-0862, Jpn 2 Deprtment of Helth nd Nutrition, Niigt University of Helth nd Welfre, 1398 Shimmi-ho, Kit-ku, Niigt 950-3198, Jpn 3 Fulty of Sport Sienes, Wsed University, 135-1 Horinouhi, Tokorozw, Sitm 359-1165, Jpn (Sumitted 26 July 2012 Finl revision reeived 9 Novemer 2012 Aepted 17 Deemer 2012 First pulished online 7 Ferury 2013) Astrt It is well known tht ingestion of protein soure is effetive in stimulting musle protein synthesis fter exerise. In ddition, there re numerous reports on the impt of leuine nd leuine-rih whey protein on musle protein synthesis nd mmmlin trget of rpmyin (mtor) signlling. However, there is only limited informtion on the effets of whey protein hydrolystes (WPH) on musle protein synthesis nd mtor signlling. The im of the present study ws to ompre the effets of WPH nd mino ids on musle protein synthesis nd the initition of trnsltion in skeletl musle during the post-exerise phse. Mle Sprgue Dwley rts swm for 2 h to depress musle protein synthesis. Immeditely fter exerise, the nimls were dministered either rohydrte (), plus n mino id mixture (AA) or plus WPH. At 1 h fter exerise, the supplements ontining whey-sed protein (AA nd WPH) used signifint inrese in the frtionl rte of protein synthesis (FSR) ompred with. WPH lso used signifint inrese in FSR ompred with AA. Post-exerise ingestion of WPH used signifint inrese in the phosphoryltion of mtor levels ompred with AA or. In ddition, WPH used greter phosphoryltion of riosoml protein S6 kinse nd eukryoti initition ftor 4E-inding protein 1 thn AA nd. In ontrst, there ws no differene in plsm mino id levels following supplementtion with either AA or WPH. These results indite tht WPH my inlude tive omponents tht re superior to mino ids for stimulting musle protein synthesis nd inititing trnsltion. Key words: Whey protein hydrolystes: Amino ids: Mmmlin trget of rpmyin: Protein synthesis Skeletl musle proteins turn over regulrly to the extent tht 1 2 % of proteins re synthesised nd roken down dily (1). The turnover of proteins involves the ongoing proesses of protein synthesis nd rekdown, nd it is well known tht prolonged exerise depresses musle protein synthesis (2,3). Previous studies hve demonstrted tht ingestion of mel ontining protein immeditely following exerise stimultes protein synthesis in skeletl musles (4,5). In ddition, leuine lone stimultes musle protein synthesis to the sme extent s omplete protein or omplete mixture of mino ids. This effet is due to the dose-dependent tivtion of severl ftors involved in the initition of mrna trnsltion, nd is medited primrily through the mmmlin trget of rpmyin (mtor) signlling pthwy. This pthwy inludes riosoml protein S6 kinse (S6K1) nd eukryoti initition ftor 4E-inding protein 1 (4E-BP1) (6,7). (8 10) Severl reserhers hve investigted the effet of different protein soures suh s whey, soy, sein nd whet on musle protein synthesis nd mtor signlling. These studies hve demonstrted tht whey protein stimultes musle protein synthesis nd initites trnsltion to greter extent thn the other protein soures. This finding led Norton et l. (9) to suggest tht the leuine ontent of mel ws proportionl to the pek tivtion of musle protein synthesis. These results indited tht the greter effet of whey protein on musle protein synthesis ws due to its high leuine ontent. Ktsnos et l. (11) lso suggested tht rul of musle protein in elderly sujets ws greter following ingestion of whey protein thn ingestion of its onstituent essentil mino id ontent. Although there is lrge volume of evidene on the effet of leuine nd leuine-rih intt whey protein on musle protein synthesis nd mtor Arevitions: 4E-BP1, eukryoti initition ftor 4E-inding protein 1; AA, mino id mixture; BCAA, rnhed-hin mino ids;, rohydrte; FSR, frtionl rte of protein synthesis; mtor, mmmlin trget of rpmyin; S6K1, riosoml protein S6 kinse; WPH, whey protein hydrolyste. * Corresponding uthor: A. Knd, fx þ81 465 37 3638, emil tushi.knd.@meiji.om
982 A. Knd et l. signlling, there is only limited informtion on the effets on these proesses of enzymtilly hydrolysed whey protein (whey protein hydrolystes; WPH) whih ontin mostly peptides. In previous study, we demonstrted tht ingestion of rohydrte () plus WPH fter glyogen-depleting exerise used signifintly greter inrese in glyogen ontent thn either whey protein or rnhed-hin mino ids (BCAA) (12). Tht study lso indited tht iotive peptides in WPH used greter inrese in musle glyogen synthesis thn BCAA. We therefore hypothesised tht WPH, whih ontins iotive peptides, my use greter reovery of musle protein synthesis fter endurne exerise. In the present study, we exmined whether ingestion of WPH used greter inrese in musle protein synthesis fter endurne exerise ompred with mixture of mino ids. We lso exmined whether WPH stimulted mtor signlling in the sme wy s leuine. Experimentl methods Animls Mle Sprgue Dwley rts with ody weights of pproximtely 150 g (CLEA Jpn, In.) were used in the study. The nimls were mintined t 23 ^ 28C, with lights on from 08.00 to 20.00 hours nd off from 20.00 to 08.00 hours, nd hd free ess to food (protein 23 6 %, ft 5 3 %, 54 4 %, sh 6 1 %, fire 2 9 % nd moisture 7 7 %; MF; Orientl Yest Compny Limited) nd wter. The study ws pproved y the Animl Committee of the Food Siene Reserh Lortory, Meiji Compny Limited, with nimls reeiving re ording to the guidelines lid down y this ommittee. Experimentl protool The nimls were limtised to swimming exerise y 3 d of pre-trining. At 1 d efore the experiment, the nimls were fed 5 g of restrited diet (MF; Orientl Yest Compny Limited). On the dy of the experiment, the nimls either remined in their ges (sedentry ontrols, n 8) or swm for 2 h, with four rts swimming simultneously in rrel filled to depth of 50 m, llowing n verge surfe re of 400 m 2 for eh niml. Wter temperture ws mintined t onstnt 358C during the swimming protool. Immeditely following exerise, one group of rts ws killed (exerise ontrols, n 8) nd the other groups (n 8 per group) were given orl dministrtion of one of three isoenergeti test solutions using sonde. These solutions ontined 44 kj in 4 ml test dose tht represented pproximtely 15 % of dily energy needs, s determined previously (4), nd inluded either in the form of 50 % gluose plus 50 % surose (), mixed mel ontining pproximtely 18 % mino id mixture (AA), or mixed mel ontining pproximtely 23 % WPH (Meiji Compny Limited) (Tles 1 nd 2). Eh test solution ws isoenergeti (1100 kj/100 ml). The AA nd WPH mels ontined equl mounts of protein, with the protein ontent of these preprtions eing mesured y the Kjeldhl method (13). The verge hin length of the peptides ws lulted s the rtio of totl N:mino N in the protein smples, whih showed tht whey protein hydrolyste hd n verge peptide length of 3 64. The mino id omposition of AA ws equivlent to tht of WPH nd ws prepred y mixing pure rystlline L-mino ids (Tle 3). At 1 h fter dministrtion, the remining rts were killed under diethyl ether nesthesi, nd the trieps musle exised nd stored t 2808C until further use. The deision to investigte musle reovery 1 h fter exerise ws sed on previous work in rodents, whih showed tht re-limenttion following exerise used n inrese in musle protein synthesis within 1 h fter refeeding (4). Administrtion of metoli trer At 15 min efore killing, olus dose (45 mg/kg ody weight, 22 5 mg/ml) of 2 H-lelled phenyllnine ([ 2 H 5 ]Phe; Cmridge Isotope Lortories) ws injeted vi the til vein for mesurement of the frtionl protein synthesis rte (FSR). At 15 min fter the injetion, the trieps musle ws exised nd frozen rpidly. The elpsed time from injetion until freezing ws reorded s the tul time for inorportion of the lelled mino id into the protein. Plsm mesurements Plsm insulin ws mesured using ommeril ELISA kit for rt insulin (Merodi AB) nd plsm free mino ids y HPLC, with pre-olumn 6-minoquinolyl-N-hydroxysuinimidyl rmte derivtistion (14). Plsm gluose ws mesured y enzymti methods using ommeril kit (Wko Pure Chemil Industries Limited). Mesurement of protein synthesis The rte of protein synthesis in individul tissues ws mesured y the inorportion of injeted [ 2 H 5 ]Phe into musle proteins. Determintion of [ 2 H 5 ]Phe enrihment in plsm nd hydrolysed musle protein smples ws rried Tle 1. Mronutrient omposition of the test solutions (g/100 g) Ft (g/100 g) Protein (g/100 g) (% energy) Ft (% energy) Protein (% energy) Energy (kj/g) (g/44 kj) Ft (g/44 kj) Protein (g/44 kj) 100 0 0 100 0 0 16 74 2 63 0 0 AA 80 0 18 00 80 0 18 00 16 74 2 16 0 0 47 WPH 82 7 17 91 81 95 0 15 17 90 16 75 2 15, 1 0 47, rohydrte; AA, mino id mixture; WPH, whey protein hydrolyste.
Whey protein hydrolystes nd protein synthesis 983 Tle 2. Mronutrient omposition of the whey protein hydrolystes (WPH) WPH out using modifition of the proedure, desried y Brk et l. (15), tht inorported LC/MS/MS systems (TQD; Wters Corportion) performed on 2 1 50 mm olumn with prtile size of 1 7 mm (ACQUITY UPLC BEH C18; Wters Corportion). Moile phse A onsisted of 5 % trifluoroeti id in Milli-Q wter, while the moile phse B onsisted of 5 % trifluoroeti id in etonitrile. The initil eluent omposition ws 100 % A, followed y n inrese to 40 % B for 9 0 min, 80 % for min nd then redution to 100 % A for 3 0 min. The totl running time ws 1 min, eluent flow ws 0 3 ml/min nd olumn temperture ws set t 408C. UV tre ws reorded t 215 nm, with the nlytes deteted using eletrospry ionistion in the positive mode. Multiple retion monitoring ws performed using hrteristi frgmenttion ions (m/z 166 19. 120 10) for Phe nd (m/z 171 19. 125 10) for [ 2 H 5 ]Phe. The prmeters for the LC/MS/MS nlysis were s follows: pillry voltge, 3000 V; soure temperture, 1208C; desolvtion temperture, 4008C; desolvtion gs flow, 849 litres/h; one gs flow, 48 litres/h; one voltge nd ollision energy, 25 V nd 15 ev, respetively. FSR, defined s the perentge of tissue protein renewed eh dy, were lulted ording to the formul: FSR ¼ðE 100Þ=ðE tþ; g/100 g Crohydrte 1 Ft 0 3 Protein 79 1 Moisture 2 2 Ash 6 4 where t is the time intervl etween injetion nd ooling of the tissue smple, expressed in d, nd E nd E re the enrihments of [ 2 H 5 ]Phe in hydrolysed tissue protein nd in musle free mino ids, respetively. Tle 3. Composition of the mino id mixture Amino ids g/100 g Al 4 90 Arg 2 79 Asx 10 91 Cys 2 42 Glx 17 65 Gly 1 89 His 2 20 Ile 4 82 Leu 17 Lys 9 77 Met 2 18 Phe 3 39 Pro 5 45 Ser 5 02 Thr 5 30 Trp 1 77 Tyr 3 46 Vl 5 01 Western lotting Musle superntnts were sujeted to Western lotting s desried previously (16). Phosphoryltion of mtor t Ser2448 ws deteted using rit nti-phospho-mtor (Ser2448; Cell Signling Tehnology) nd expressed s rtio of totl mtor expression, determined using ntimtor (Cell Signling Tehnology). Phosphoryltion of 4E-BP1 t Thr37/46 ws deteted using rit ntiphospho-4e-bp1 (Thr37/46; Cell Signling Tehnology) nd expressed s rtio of totl 4E-BP1 expression, determined using nti-4e-bp1 (Cell Signling Tehnology). Phosphoryltion of S6K1 t Thr389 nd (Thr421/Ser424) ws deteted using rit nti-phospho-s6k1 (Thr389; Cell Signling Tehnology) nd rit nti-phospho-s6k1 (Thr421/ Ser424; Snt Cruz Biotehnology), respetively. Phosphoryltion of S6K1 ws then expressed s rtio of totl S6K1 expression, determined using nti-s6k1 (Snt Cruz Biotehnology). Sttistil nlysis Dt re expressed s mens with their stndrd errors. All the sttistil nlyses were performed using SPSS for Windows, version 14.0J (SPSS Jpn). Comprison of dt ws rried out using one-wy ANOVA, nd when signifint differene in vrile ws deteted, the groups were ompred using Tukey s post ho nlysis. Differenes etween the groups were onsidered to e sttistilly signifint t P, 5. Assoitions etween the vriles were nlysed using Person s orreltion oeffiients. Differenes etween the groups were onsidered to e sttistilly signifint t P,5. Results Frtionl rtes of protein synthesis A 2 h period of swimming (exerise ontrols) redued FSR y 42 % ompred with the sedentry ontrol group. Feeding of lone did not promote the reovery of FSR. In ontrst, supplements ontining whey-sed protein reversed the derese in FSR. Furthermore, WPH used signifint inreses in FSR ompred with AA or (Fig. 1). Plsm insulin nd mino id levels Plsm insulin levels were higher in the WPH group thn in the group (Tle 4). However, there ws no signifint differene in insulin levels etween the AA nd WPH groups. Ingestion of whey-sed protein used greter inrese in plsm onentrtions of leuine, totl mino ids, essentil mino ids nd BCAA ompred with (Tle 4), wheres there ws no signifint differene in these levels etween the AA nd WPH groups. Plsm gluose levels did not differ etween the groups.
984 A. Knd et l. Skeletl musle FSR (%/d) 8 6 4 2 0 Phosphorylted mmmlin trget of rpmyin, eukryoti initition ftor 4E-inding protein 1 nd riosoml protein S6 kinse levels Post-exerise ingestion of gluose plus WPH used signifint inrese in phosphorylted mtor levels ompred with AA or (Fig. 2(A)). The WAA group lso showed signifint inrese in phosphorylted mtor ompred with the group. The WPH group hd greter phosphoryltion of 4E-BP1 ompred with oth the AA nd groups (Fig. 2(B)). The AA group lso exhiited signifint inrese in phosphorylted 4E-BP1 ompred with the group. Phosphoryltion of S6K1 t Thr389 nd Thr421/Ser424 ws greter in the WPH group thn in the AA nd groups (Fig. 2(C) nd (D), respetively). The AA group lso demonstrted greter phosphoryltion of S6K1 t oth these sites ompred with the group. Disussion SED EX Fig. 1. Frtionl rtes of protein synthesis (FSR) in rts dministered diets tht differed in moleulr forms of dietry whey-sed protein, mesured 1 h fter swimming tivity. Vlues re mens (n 8), with their stndrd errors represented y vertil rs.,, Men vlues with unlike letters were signifintly different (P,5; Tukey s post ho nlysis). SED, sedentry ontrol; EX, exerise ontrol;, rohydrte; AA, mino id mixture; WPH, whey protein hydrolyste. In the present study, we ompred the effet of different moleulr forms of dietry protein on the reovery of musle protein synthesis nd mtor signlling fter endurne exerise. We exerised rts using 2 h of swimming to depress musle protein synthesis. Reovery of musle protein synthesis ws stimulted y whey-sed protein (AA or WPH) supplementtion. Furthermore, the WPH group showed signifintly higher musle protein synthesis ompred with the AA group. This is the first study to demonstrte tht WPH is more effetive in the reovery of skeletl musle protein synthesis thn mixture of whey mino ids, when given s the protein soure fter exerise. Furthermore, the present results suggest tht it is possile tht ertin iotive peptides ontined in WPH re superior to mino ids for stimulting musle protein synthesis. It is well known tht BCAA, espeilly leuine, ply n importnt role in the tivtion of musle protein synthesis (17,18). Leuine lone or s supplement hs een shown to stimulte musle protein synthesis fter exerise in models of oth endurne nd resistne exerise (19,20), leding Norton et l. (9) to suggest tht pek tivtion of musle protein synthesis ws proportionl to the leuine ontent of mel. On the other hnd, the mino id ompositions of the two solutions used in our study (WPH nd AA) were equivlent, nd therefore totl BCAA nd leuine intke ws equl. In ddition, plsm mino id levels did not differ etween the two groups, lthough the effets of the two diets on musle protein synthesis vried. This result strongly suggests tht not only the leuine ontent in dietry protein ut lso the moleulr form of dietry protein soures my e importnt determinnts of musle protein synthesis. Furthermore, we onlude tht WPH used in the present study ontined tive omponents tht re superior to leuine itself for inresing musle protein synthesis. Reently, we demonstrted tht BCAA-ontining peptides in WPH, suh s Ile-Vl, Leu-Vl, Vl-Leu, Ile-Ile, Ile-Leu, Leu-Ile nd Leu-Leu, inresed the rte of gluose uptke in isolted rt skeletl musle. This finding suggested tht these BCAAontining peptides re iotive peptides tht promote musle glyogen synthesis (21). Furthermore, we hve shown tht the plsm levels of these peptides were elevted mrkedly fter ingestion of WPH (14). It is therefore possile tht BCAA-ontining peptides my t s iotive peptides in oth glyogen nd musle protein synthesis in skeletl musle. Tle 4. Plsm mino id nd insulin levels of rts dministered diets tht differed in the moleulr forms of dietry whey-sed protein, mesured 1 h fter swimming (Men vlues with their stndrd errors) Men SE Men SE Men SE Leu (mmol/l) 176 6 16 9 409 8 44 1 423 9 28 8 TAA (mmol/l) 4949 0 159 2 7368 3 420 2 7119 8 208 0 EAA (mmol/l) 1467 4 79 5 2577 6 232 7 2584 7 128 6 BCAA (mmol/l) 552 8 49 4 1115 6 111 8 1133 8 73 0 Gluose (mmol/l) 1 9 3 0 7 9 2 0 6 Insulin (pmol/l) 76 6 9 7 107 9, 20 1 147 9 20 4, rohydrte; AA, mino id mixture; WPH, whey protein hydrolyste; TAA, totl mino ids; EAA, essentil mino ids; BCAA, rnhed-hin mino ids., Men vlues within row with unlike supersript letters were signifintly different (P,5; Tukey s post ho nlysis).
Whey protein hydrolystes nd protein synthesis 985 (A) (B) p-(thr37/46) p-ser2448 mtor 4E-BP1 p-ser2448:totl mtor (ritrry units) 2 5 AA WPH p-(thr37/46):totl 4E-BP1 (ritrry units) (C) (D) p-(thr421/ Ser424) S6K1 p-thr389 S6K1 p-thr389:totl S6K1 (ritrry units) 2 5 mtor signlling is key regultory ftor for musle protein synthesis nd is regulted positively y mino ids, prtiulrly leuine (22 24). In the present study, ingestion of WPH used signifintly greter phosphoryltion of mtor levels ompred with mixture of mino ids, despite the plsm levels of mino ids in AA nd WPH eing similr. Tken together, these results indited tht tive omponents, suh s iotive peptides, ontined in WPH hve greter effet on mtor phosphoryltion thn leuine degrded from WPH. It is lso known tht two well-studied downstrem trgets of mtor, S6K1 nd 4E-BP1, influene trnsltion initition nd elongtion (25,26). In the present study, we demonstrted tht phosphoryltion of S6K1 nd 4E-BP1 ws higher in the WPH group thn in the AA group. These results indite tht WPH used greter phosphoryltion of S6K1 nd 4E-BP1 vi mtor tivtion thn AA. Krlsson et l. (27) lso demonstrted tht BCAA inrese the phosphoryltion of S6K1 t Thr421/Ser424 without tivting mtor (28). Tken together, these erlier findings nd the present results suggest tht ertin iotive peptides in WPH my lso tivte S6K1 vi phosphoryltion of Thr421/ Ser424 without tivting mtor. We lso showed tht plsm levels of dipeptides were elevted mrkedly fter ingestion of WPH (14). However, the plsm levels of dipeptides were lower thn those of plsm p-(thr421/ser424):totl S6K1 (ritrry units) Fig. 2. Skeletl musle phosphorylted (A) mmmlin trget of rpmyin (mtor), (B) eukryoti initition ftor 4E-inding protein 1 (4E-BP1), riosoml protein S6 kinse (S6K1) nd (C) Thr389 nd (D) Thr421/Ser424 levels of rts dministered diets tht differed in moleulr forms of dietry whey-sed protein, mesured 1 h fter swimming. Phosphorylted mtor ws normlised for totl mtor, phosphorylted 4E-BP1 for totl 4E-BP1 nd phosphorylted S6K1 for totl S6K1. Vlues re mens (n 8), with their stndrd errors represented y vertil rs.,, Men vlues with unlike letters were signifintly different (P,5; Tukey s post ho nlysis)., rohydrte; AA, mino id mixture; WPH, whey protein hydrolyste. mino ids. In the present study, plsm leuine levels in the AA nd WPH groups were 409 8 nd 423 9 mmol/l, respetively. In ontrst, plsm dipeptide levels, whih re sed on WPH, were extremely low ompred with leuine levels. For exmple, the plsm levels of Ile-Vl, Leu-Vl, Vl-Leu, Ile-Ile, Leu-Ile, Ile-Leu nd Leu-Leu were 11 1 (SE 1 4), 6 0 (SE 0 7), 25 9 (SE 3 1), 9 4 (SE 1 1), 1 7 (SE 0 2), 13 2 (SE 1 3) nd 34 2 (SE 4 3) nmol/l, respetively, in the WPH group (A Knd, K Nkym, T Fuksw, J Kog nd K Kwnk, unpulished results). On the other hnd, these dipeptides were not deteted in the AA group. Additionl studies re therefore required to determine whether these dipeptides used stimultion of mtor signlling t lower plsm onentrtions thn leuine. Previous studies (29) hve indited tht miniml onentrtion of plsm insulin is required for oth mino id- nd exerise-indued stimultion of trnsltion initition nd protein synthesis in skeletl musle. In prtiulr, insulin vilility is importnt in mtor-medited signlling. We hve shown previously tht the plsm insulin response during the 0 60 min period ws higher for protein hydrolystes thn for whole protein (14). In the present study, the WPH group hd higher plsm insulin levels thn the AA group, lthough this differene ws not sttistilly signifint. We lso oserved signifint orreltion etween insulin nd
986 A. Knd et l. oth FSR (r 1, P¼12) nd phosphorylted mtor levels (r 0 66, P, 01). Tken together, these findings suggest tht greter inrese in plsm insulin levels used y WPH my indue lrger inrese in FSR vi tivtion of mtor signlling ompred with AA. It is well known tht swimming exerise is highly stressful tivity tht results in n inresed relese of gluoortioids (30). The ssoition etween gluoortioid exess nd musle trophy is well estlished (31). Inhiition of protein synthesis hs een oserved onsistently in gluoortioid-treted nimls (32). In ddition, the potent syntheti gluoortioid dexmethsone lso prevents mino id-indued hyperphosphoryltion of S6K1 nd 4E-BP1, s well s stimulting protein synthesis in musle (33). It is therefore possile tht WPH used n inrese in musle protein synthesis not only y tivting mtor signlling, ut lso y ttenuting the effet of gluoortioids. Further studies re therefore required to investigte the effets of WPH on gluoortioids. In onlusion, the present results demonstrte tht ingestion of WPH inreses musle protein synthesis to greter extent thn mixture of mino ids fter endurne exerise s result of greter phosphoryltion of mtor levels. These results indited tht WPH my ontin tive omponents, suh s iotive peptides, tht re superior to leuine for stimulting mtor signlling. The findings of the present study therefore provide new insights into the effets of different forms of dietry protein for sports nutrition. Aknowledgements No funding ws reeived for the present study. A. K. nd K. N. designed the reserh; A. K., T. F., J. K., M. K., K. K. nd M. H. onduted the reserh; A. K. nlysed the dt, wrote the pper nd hd primry responsiility for the finl ontent. All uthors red nd pproved the finl mnusript. The uthors hve no onflits of interest to delre. Referenes 1. Welle S, Thornton C, Sttt M, et l. (1994) Postprndil myofirillr nd whole ody protein synthesis in young nd old humn sujets. Am J Physiol 267, E599 E604. 2. 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