rtiles Integrtive Physiology nture pulishing group Dietry Whey Protein Dereses Food Intke nd Body Ft in Rts June Zhou 1 3, Mihel J. Keenn 2, Jk N. Losso 2, Anne M. Rggio 1,2, Li Shen 1,2, Kthleen L. MCutheon 2, Rihrd T. Tulley 2, Mr R. Blkmn 3 nd Roy J. Mrtin 1,2 We investigted the effets of dietry whey protein on food intke, ody ft, nd ody weight gin in rts. Adult (11 12 week) mle Sprgue Dwley rts were divided into three dietry tretment groups for 1-week study: ontrol. Whey protein (), or high-protein ontent ontrol (). Alumin ws used s the si protein soure for ll three diets. nd diets ontined n dditionl 24% (wt/wt) whey or isoflvone-free soy protein, respetively. Food intke, ody weight, ody ft, respirtory quotient (RQ), plsm holeystokinin (CCK), glugon like peptide-1 (GLP-1), peptide YY (PYY), nd leptin were mesured during nd/or t the end of the study. The results showed tht ody ft nd ody weight gin were lower (P <.5) t the end of study in rts fed or vs. ontrol diet. The umultive food intke mesured over the 1-week study period ws lower in the vs. ontrol nd groups (P <.1). Further, fed rts exhiited lower N 2 free RQ vlues thn did ontrol nd groups (P <.1). Plsm onentrtions of totl GLP-1 were higher in nd vs. ontrol group (P <.5), wheres plsm CCK, PYY, nd leptin did not differ mong the three groups. In onlusion, lthough dietry nd eh derese ody ft umultion nd ody weight gin, the mehnism(s) involved pper to e different. fed rts exhiit inresed ft oxidtion, wheres fed rts show deresed food intke nd inresed ft oxidtion, whih my ontriute to the effets of whey protein on ody ft. Oesity (211) 19, 1568 1573. doi:1.138/oy.211.14 Introdution Whey protein is n undnt yprodut in heese prodution (1 liter of milk produes out 9 liter of whey during heese mnufture (1). Beuse it ontins ll essentil mino ids nd n rpidly inrese plsm mino id onentrtions, whey protein is onsidered to e high qulity protein (2,3), nd is thus ommonly used for mintining helthy ody weight in humns (4 6). Although, in generl, high-protein diet dereses food intke (7 1), whey protein shows greter suppression of ppetite in the next hour of feeding thn do egg-lumin, soy protein, nd sein (5,11,12). Whey protein is lso more effetive thn red met in reduing ody weight gin nd inresing insulin sensitivity (6). These studies indite tht my hve unique properties in mintining helthy ody weight. However, most studies on whey protein suppression of ppetite in humns hve een shortterm, with food intke or stition mesured severl hours fter onsumption of single whey protein mel. The results re lso ontrditory euse the durtion to the next mel nd omposition of the test mels were vried in these studies (5,11 13). Thus, longer term studies nd use of refully mthed dietry omponents for test nd ontrol diets re needed to lrify the role of whey protein on food intke nd ody weight. In this study, we used rt model nd refully designed ontrol diets to exmine the longer term effets of dietry whey protein on ody ft nd food intke. During 1 week study period, food intke, ody ft, ody weight gin, nd metoli rte (respirtory quotient (RQ)) were mesured in rts fed whey protein or two different ontrol diets. There is onsiderle interest in whether whey protein ffets stiety nd food intke vi ltertions in the seretion or tion(s) of the gut sereted noreti hormones, glugon like peptide-1 (GLP-1), peptide YY (PYY), nd holeystokinin (CCK) (14 16). Thus, we mesured plsm onentrtions of GLP-1, PYY, nd CCK. Leptin, nother importnt hormone in regultion of food intke nd ody ft, ws lso mesured. Two ontrol diets were used in this study: one hd the protein ontent of the AIN-93M sed rodent diet (14%), wheres the other hd the sme higher protein ontent (38%) s the whey protein diet, lthough nother soure of protein ws used. This study design llowed us to exmine the effets of dietry whey protein vs. high dietry protein in generl, on food intke nd ody ft. 1 Pennington Biomedil Reserh Center, Louisin Stte University System, Bton Rouge, Louisin, USA; 2 Louisin Stte University Agriulturl Center, Bton Rouge, Louisin, USA; 3 Reserh Servie, Veterns Affirs Medil Center, Wshington, DC, USA. Correspondene: June Zhou (june.zhou@v.gov) Reeived 1 Septemer 21; epted 8 Jnury 211; pulished online 17 Ferury 211. doi:1.138/oy.211.14 1568 VOLUME 19 NUMBER 8 ugust 211 www.oesityjournl.org
rtiles Integrtive Physiology Methods nd Proedures Animls nd housing onditions Thirty helthy dult mle Sprgue Dwley rts (11 12 week old) were purhsed from Hrln Industries (Indinpolis, IN). After qurntine, the rts were single housed in suspended wire-ottom ges in humidity nd temperture ontrolled room (22 ± 2 C, 65 67% humidity) on 12:12 h light:drk yle with free ess to food nd wter. Wire-ottom ges were used to mesure food spillge. All rts were fed semipurified powder diets prepred in our lortory. Rts were fed the AIN-93M sed rodent ontrol diet nd limted to experimentl onditions until their ody weights were stle (out 2 weeks). The protool ws pproved y the Pennington Biomedil Reserh Center s Animl Cre nd Use Committee. Diets The regulr ontrol (ontrol) diet ws prepred sed on the AIN-93M formul for lortory rodents (17) nd egg lumin ws used s the protein soure. The omponents of the whey protein () diet were the sme s those of the ontrol diet, exept tht hlf the strh in the diet ws repled y whey protein. The high-protein ontent ontrol () diet hd the sme protein ontent s the diet, lthough the whey protein ws repled y isoflvone-free soy protein. This diet ws designed to ompre whey protein with non-milk protein soure. Isoflvone-free soy protein ws used to void the reported iotivities of isoflvones tht re nturlly ssoited with soy protein. The totl protein ontents were 14% for diet, 38% for diet (14% egg lumin plus 24% ), nd 38% for diet (14% egg lumin plus 24% isoflvone-free soy protein). The detiled ompositions of the three experimentl diets re listed in Tle 1. Tle 1 Experimentl diets omponents Ingredients (g) diet diet diet Alumin (egg white) 14 14 14 Whey protein onentrte 8 3 Soy protein 353.74 Strh 612.5 312.5 258.8 Surose 1 1 1 Soyen oil 4 4 4 Cellulose 5 5 5 Minerl mix for egg white protein 35 35 35 Vitmin mix 1 1 1 Choline hloride 2.5 2.5 2.5 Biotin/Surose Premix 1 1 1 Totl (g) 1, 1, 1, BHT (in oil).8.8.8 Totl protein ontent (%) 14 38 38 Totl ft ontent (%) 4 4 4 Totl rohydrte ontent (%) 82 58 58 The unit for ll ingredients is grm. Whey protein (whey protein onentrte 8, gift from the Diry Counil) ontins 79.7% whey protein. Isoflvone-free soy protein (PROCON 2, gift from The Sole Compny, St Louis, MO) hs 67.6% soy protein. Strh (1% mylopetin) ws gift from Ntionl Strh Food Innovtion (Bridgewter, NJ 887) All other ingredients were purhsed from Dyets (Bethlehem, PA). The energy ontent of the three experimentl diets were similr (3.6 3.7 kl/ kg diet)., high-protein ontent ontrol;, whey protein; BHT, utylted hydroxytoluene. Experimentl protool After limtiztion to the experimentl onditions (powder diet nd wire ges), rts were divided into three dietry tretment groups for 1 week study. Eh group hd similr verge ody weight nd ody ft t the eginning of the study. Fresh food ws provided to eh rt three times per week y hnging the food jr of eh rt. Food intke ws reorded y weighing the food jr efore it ws pled into the ge with fresh diet (full jr weight) nd when it ws tken out of the ge (empty jr weight) 2 or 3 dys lter. Food spillge ws lso reorded y refully weighing the spillge under the wire ge for eh rt. Beuse ll three diets were powder diets, there were no differenes in diet onsisteny tht would hve mde the spillge of one diet hrder to mesure thn tht of the other diets. The food intke ws lulted y sutrting the weights of the empty food jrs nd spillge from the weights of the full food jrs for eh rt. Body weight ws lso mesured three times per week when food intke ws mesured. During the first 6 weeks of the study period, rts were undistured exept for food intke nd ody weight mesurements. At the 6th week of the study, ody omposition ws mesured y nuler mgneti resonne (NMR) in ll rts. On the 7th nd 8th weeks of the study, rts were pled in metoli hmers for mesurement of respirtory exhnge rtios (RQ s). Body weight gin ws lulted s the ody weight on the dy of mesurement minus the ody weight reorded t the eginning of study (dy mesurement dy ). All rts were killed y depittion t the end of the study. Trunk lood ws olleted from eh rt for mesurements of GLP-1, PYY, CCK, nd leptin. Body ft nd len ody mss mesurements during the study Body ft nd len ody mss were mesured during the study y NMR (Bruker minispe Live Rts Anlyzer model mq7.5, LF9; Bruker Optis, Woodlnds, TX) (18). Eh rt ws weighed efore it ws pled in the tue for NMR mesurement nd the reorded ody weight ws used for ody omposition lultion. For eh rt, the mesurements were repeted twie nd the differene of % ft for eh rt ws <3%. If not, third mesurement ws otined nd the two loser vlues (within 3% differene) were verged nd used for lultion of ody ft nd len ody mss. The % totl ody ft or % len ody mss ws lulted s the ft mss or len ody mss divided y ody weight. Body ft mesurement t the end of experiment Totl dominl ft pds (retroperitonel, perirenl, nd epididyml) were removed nd wet weights were mesured on n nlytil lne. Body weight ws reorded efore the ft dissetion ws performed. The totl dominl ft ws onsidered to e the sum of three ft pds, nd % ody ft ws lulted s totl dominl ft divided y ody weight. Indiret lorimetry Rts were pled in metoli hmers (Oxymx; Columus Instruments, Columus, OH) t room temperture (23 C) for 6 dys. The first 3 dys served s n dpttion period. Oxygen onsumption (VO 2 ), ron dioxide expirtion (VCO 2 ), nd respirtory exhnge rte (RQ: VCO 2 / VO 2 ) were reorded ontinuously during the lst 3 dys. Dt olleted from the lst 2 dys of the 3 dy dt olletion period were used for nlysis. Beuse the diet nd HP diet hd signifintly higher mounts of dietry protein thn did the ontrol diet, (38% vs. 14%), N-free RQ ws lulted (19) nd used s the inditor of metoli rte. GLP-1, PYY, CCK, nd leptin mesurements At the end of the study, trunk lood from eh rt ws olleted in EDTA tues nd entrifuged t 4,g for 2 min to extrt plsm. Plsm totl GLP-1, PYY, CCK, nd leptin were mesured y rdioimmunossy with kits from Lino Reserh (St Louis, MO). The intr-ssy oeffiients of vritions were within 9.7% for GLP-1, 7.8% for PYY, 5.5% for CCK, nd 9.4% for leptin. The interssy oeffiient of vrition is not pplile euse eh hormone ws mesured within single ssy. oesity VOLUME 19 NUMBER 8 August 211 1569
rtiles Integrtive Physiology Sttistil nlysis Dt re presented s men vlues ± s.e.m. One-wy ANOVA followed y n F-proteted lest signifint differene method ws used to nlyze ll dt. Body weight gin dt were ompred within the sme dy for the three groups of rts. For the umultive food intke, dt were nlyzed using repeted mesures ANOVA with diet s the independent vrile. Food intke t the strt of the experiment ws used s ovrite in the nlysis. Post-ho omprisons of tretment mens t different time points were exmined y t-test nd the signifine levels reported were undjusted for multiple omprisons. For ll tests desried ove, P <.5 ws onsidered signifint. SAS (SAS for Windows, Relese 9.2; SAS Institute, Cry, NC) ws used for dt nlysis. Results Body weight Body weight gin during the experimentl period is shown in Figure 1. Both the group nd group hd lower ody weight gins thn did the ontrol group t the end of the experiment (P <.5). Body weight gin (g) 9 8 7 6 5 4 3 2 1 1 2 3 4 5 6 7 8 9 Weeks on experimentl diet Figure 1 Body weight gin for rts fed ontrol,, or diets. Dt represent men vlues ± s.e.m. (n = 1 11 per group). Letters elow the lines indite signifint differenes (P <.5), s shown. () group is lower thn oth ontrol nd groups. () is lower thn ontrol, nd is etween the nd ontrol ut not different from either of them. () Both nd re lower thn ontrol., high-protein ontent ontrol;, whey protein. Cumultive food intke (g) 1,2 1, 8 6 4 2 1 2 3 4 5 6 7 8 9 Weeks on diet Figure 2 Cumultive food intke for rts fed ontrol,, or diets for 1 weeks. Dt represent men vlues ± s.e.m. (n = 1 11 per group). Indites signifint differene (P <.1) ompred with ontrol or groups., high-protein ontent ontrol;, whey protein. Food intke Cumultive food intke is shown in Figure 2. The umultive food intke in fed rts ws signifintly lower thn in oth ontrol nd groups from week 5 until the end of the study (P <.1). Body ft nd len ody mss Body ft mesured y NMR during the 6th week of the study (Figure 3) ws lower in the fed rts thn in the diet group (P <.5). Body ft of ontrol diet fed rts ws intermedite etween, nd did not differ signifintly from tht of either the or groups. There ws no signifint differene for len ody mss mong three groups, determined from the sme NMR mesurement (dt not shown). At the end of the study, ody ft in the two high-protein diet groups ws lower thn tht of the ontrol diet fed rts (P <.5, Figure 3). Metoli rte The N-free RQ vlues differed signifintly (P <.1) mong ll three tretment groups (Figure 4), with the lowest vlues in % Totl ody ft/ody weight 18 17 16 15 14 % Adominl ft/ody weight 3. 2.5 2. 1.5 1..5. Figure 3 Body ft mesured t () week 6 y nuler mgneti resonne nd () t the end of study y dissetion for rts fed ontrol,, or diets. () Body ft (%) ws lulted s totl ody ft divided y ody weight () or the sum of dominl ft pds divided y ody weight. Dt represent men vlues ± s.e.m. (n = 9 11 per group). Letters ove the olumns indite signifint differenes (P <.5)., high-protein ontent ontrol;, whey protein. N-free RQ (48 hours men) 1.9.8.7 Light Drk Totl Figure 4 N-free respirtory quotient (light yle, drk yle, totl light + drk yle) of rts fed ontrol,, or diets for 7 8 weeks. The detiled lultion method is desried in the mnusript. Dt represent men vlues ± s.e.m. (n = 1 11 per group). Letters ove the olumns indite signifint differenes within the time period (P <.1)., high-protein ontent ontrol;, whey protein. 157 VOLUME 19 NUMBER 8 ugust 211 www.oesityjournl.org
rtiles Integrtive Physiology 5 4 Totl GLP-1 2.5 2. CCK 2. 1.6 Leptin 1 8 PYY pm 3 2 pmol/l 1.5 1. ng/ml 1.2.8 pg/ml 6 4 1.5.4 2.. Figure 5 Plsm hormones mesured y rdioimmunossy t the end of study for rts fed ontrol,, or diets. Dt represent men vlues ± s.e.m. (n = 9 11 per group). Different letters ove the olumn indite signifint differenes (P <.1). CCK, holeystokinin;, highprotein ontent ontrol;, whey protein; GLP-1, glugon like peptide-1; PPY, peptide YY. the group, intermedite vlues in the group, nd the highest vlues in the ontrol group. Hormone mesurements Hormones were mesured in the plsm olleted t the end of the study (Figure 5). Plsm totl GLP-1 onentrtions were higher in the nd groups ompred with the ontrol diet group (P <.1). There were no signifint differenes in leptin, CCK, nd PYY onentrtions mong the three groups. Disussion The purpose of this study ws to ompre the effets of whey protein with non whey protein on food intke nd ody ft. We hose isoflvone-free soy protein s the omprle protein soure, s it is non-milk sed protein soure nd is widely used in mintining helth ody weight. In ddition, we used isoflvone-free soy protein to eliminte the potentil effets of estrogeni tivity of soy (2) on food intke (21). Our results demonstrte tht high-protein diet (38% protein ontent) using either whey or isoflvone-free soy protein redues ody weight gin, ody ft nd N-free RQ in rts ompred to the ontrol diet. However, the mehnisms involved my differ, sed on the dietry protein used. The redution of N-free RQ ws greter in vs. fed rts, nd the redution of umultive food intke ws only oserved in fed rts. The ppetite suppressive effet of is ontroversil sed on previous pulitions. In severl short-term humn studies (11,12,22 26), whey protein mel ws ompred with ontrol mels tht hd lower protein ontent nd other mels tht hd similr protein ontent s the whey mel. The protools of those studies were similr. Severl hours fter ingestion of single mel (either whey or other test mel), the sujet ws provided with stndrd mel, nd the ppetite suppressive effet ws judged y mesuring intke of the susequent mel. These protools were designed to exmine hnges in postprndil stiety hormone seretion. Thus, the susequent mesurements for food intke were short-term. Those studies onsistently demonstrted the ppetite suppressive effet of whey, ompred with ontrol mel. But results vried when the whey mel ws ompred with test mels tht hd equl protein ontent. None of those studies reported 24 h or longer term food intke. The effet of whey protein on food intke hs lso een ompred with test mels of equl protein ontent t two different totl dietry protein levels: 1% or 25%. Whey protein suppressed susequent food intke of the next mel t low protein onentrtion (1% of dietry energy), ut not t the higher protein onentrtion (25% of dietry energy) (27). In niml models, dietry whey protein suppressed food intke more thn did soy or gluten during 7 dy period (28). In ontrst, dietry whey protein did not signifintly redue verge dily food intke in high ft indued oese rts in n 8 week study (29). There re two mjor differenes when ompring this study with those ited ove. First, we did not investigte hourly or dily food intke ltertions fter whey protein ingestion. Seondly, the food intke ws reported s umultive food intke over the entire study period. The umultive food intke in fed rts ws onsistently lower thn tht in ontrol nd groups. This smll redution rehed sttistil signifine t the 5th week of the study nd ontinued to the end of the study. We used umultive food intke s the inditor of food intke, s it represents smll, ut onsistent, hnge in food intke over long period of time. Suh dt re diffiult to otin urtely in humn studies. This mild, ut onsistent, redution of food intke suggests tht whey protein regultes food intke vi distintly different mehnism thn in fed rts. The high-protein ontent nd ltertion in GLP-1 re unlikely ssoited with this unique mehnism diretly, s the group exhiited the sme hnges in noreti hormone profile s the group, ut did not show ny redution of food intke. The unique mino id omposition in whey protein might e involved in the mehnism of food intke redution. Whey protein, ompred to the other protein soures, ontins the highest onentrtion of rnhed-hin mino ids, espeilly L-leuine (3). Leuine n enter the rin from the lood more rpidly thn other mino ids (31). We hve reported tht leuine is importnt in ontrolling food intke nd n derese 24 h food intke in rts. This hypothlmi tion is relted to regultion of mrna expression of gouti-relted peptide, long oesity VOLUME 19 NUMBER 8 August 211 1571
rtiles Integrtive Physiology term regultor of food intke (32). Therefore, dietry whey protein might derese food intke through its speil mino id profile, espeilly leuine, nd ltering neuropeptide synthesis in the hypothlmus, n importnt site for ontrolling food intke t the rin. In this study, the three experimentl diets hd different protein ontents tht might hve ffeted urinry N loss. Thus, N-free RQ ws used for lultion of metoli rtes for rts in three different dietry groups. We deteted deresed RQ in oth the nd groups ompred with the regulr ontrol group. This derese in RQ is in greement with the deresed ody ft nd ody weight gin in rts fed oth nd diets, nd might hve resulted from generl effets of the high-protein ontent in the diet. However, rts in the protein group hd lower RQ thn did rts in the group. Our result is onsistent with the oservtion from reently pulished humn study wherein whey protein showed deresed RQ ompred to soy protein (13). Thus, it is possile tht whey protein is more effetive thn soy protein in deresing RQ in rts. One limittion of this study is tht the ext totl len nd ft mss of rts were not known when indiret lorimetry ws onduted, or t the end of study. Thus, we ould not speify the reltive ontriution of musle vs. ft mss to deresed RQ for nd fed rts. Although protein in generl, nd whey protein in prtiulr, n stimulte postprndil GLP-1, PYY, nd CCK seretion in humns (11,33), we deteted no signifint differenes in plsm onentrtions of PYY, nd CCK mong the three dietry tretment groups of rts. Of note, we re interested in longer term, rther thn the mel effets, of whey protein on gut hormone seretion. Thus, we did not mesure the postprndil levels of those hormones. Blood smples were olleted in the morning, nd most food intke ourred during the previous evening (round 6 PM when lights were turned off). As result, hormone levels in the morning were ffeted less y the mel relted flutution. This my explin the differene oserved etween this study nd previously reported humn studies (11,33). Similrly, elevted levels of totl GLP-1 in oth protein nd groups re likely independent of the postprndil effet of diets. The mehnism for the inresed GLP-1 remins to e lrified. Although totl GLP-1 ws inresed in the group, it is unlikely to hve influened food intke in our study. First, the group exhiited similrly elevted GLP-1 vlues, nd food intke ws not deresed in this group. Seond, reent pulitions suggest tht GLP-1 prtiiptes more in gluose ontrol thn in redution of food intke (34,35). Nevertheless, the elevted plsm GLP-1 is ssoited with higher dietry protein ontent nd lower N-free RQ in the nd vs. ontrol groups. It hs een suggested tht fsting plsm GLP-1 levels re diretly relted to n inresed ft oxidtion rte in humns (36,37). Thus, stimultion of GLP-1 seretion might e involved in ody ft redution y high-protein diet in generl in this study. In onlusion, rts fed high-protein diets enrihed with whey protein or isoflvone-free soy protein for ten weeks exhiited lower ody ft nd ody weight gins thn did ontrol rts. fed rts exhiited inresed ft oxidtion, wheres fed rts hd deresed food intke nd greter inrese in ft oxidtion, whih my hve ontriuted to the effets of whey protein on ody ft. Aknowledgments This study ws supported y Ntionl Diry Counil ontrt (numer: 1446) to Drs J.Z. nd R.M. This pper ws pproved for pulition y the Diretor of the Louisin Agriulturl Experiment Sttion s mnusript numer 21-239-9617. Dislosure The uthors delred no onflit of interest. 211 The Oesity Soiety Referenes 1. Mnso MA, Lopez-Fndino R. κ-csein mropeptides from heese whey: physiohemil, iologil, nutritionl, nd tehnologil fetures for possile uses. Food Rev Int 24;2:329 356. 2. H E, Zemel MB. Funtionl properties of whey, whey omponents, nd essentil mino ids: mehnisms underlying helth enefits for tive people (review). J Nutr Biohem 23;14:251 258. 3. 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Reeves PG, Nielsen FH, Fhey GC Jr. AIN-93 purified diets for lortory rodents: finl report of the Amerin Institute of Nutrition d ho writing ommittee on the reformultion of the AIN-76A rodent diet. J Nutr 1993;123:1939 1951. 18. Tiher GZ, Tinsley FC, Reidermn A, Heimn ML. Quntittive mgneti resonne (QMR) method for one nd whole-ody-omposition nlysis. Anl Bionl Chem 23;377:99 12. 19. Kleier M. The Fire of Life: An Introdution to Animl Energetis. Wiley & Sons, New York, 1961. 2. de Lim Tofondo Vieir M, Durte RF, Cmpos LM, Nunn Ede A. Comprison of the estrogeni potenies of stndrdized soy extrts y immture rt uterotrophi iossy. Phytomediine 28;15:31 37. 1572 VOLUME 19 NUMBER 8 ugust 211 www.oesityjournl.org
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