Ancah Caesarina Novi Marchianti Emi Arimura Miharu Ushikai Masahisa Horiuchi

Transcription

1 Environ Helth Prev Med (214) 19: DOI 1.17/s z REGULAR ARTICLE Voluntry exerise under food restrition ondition dereses blood brnhed-hin mino id levels, in ddition to improvement of gluose nd lipid metbolism, in db mie, niml model of type 2 dibetes Anh Cesrin Novi Mrhinti Emi Arimur Mihru Ushiki Mshis Horiuhi Reeived: 14 My 214 / Aepted: 13 July 214 / Published online: 2 August 214 Ó The Jpnese Soiety for Hygiene 214 Abstrt Objetives Exerise is effetive for preventing the onset nd development of type 2 dibetes mellitus (T2DM) in humn ses; however, the effet of exerise on the pthophysiology using niml models of T2DM hs not been fully evluted. Methods We pplied voluntry exerise under pir-fed (P) onditions in db mie, n niml model of T2DM. Exerising (Ex) nd (Se) mie were pled in ge, equipped with free or loked running wheel, for 4 weeks, respetively. The mount of food onsumed by d libitum-fed wild-type mie under the Se ondition (d- WT) ws supplied to ll mie, exept d libitum db mie (d-db). Blood prmeters nd expression of the genes involved in nutrient metbolism were nlyzed. Results PEx-db (pir-fed nd exerising) mie showed signifintly lower HbA1, body weight nd liver weight Eletroni supplementry mteril The online version of this rtile (doi:1.17/s z) ontins supplementry mteril, whih is vilble to uthorized users. A. C. N. Mrhinti E. Arimur M. Ushiki M. Horiuhi (&) Deprtment of Hygiene nd Helth Promotion Mediine, Kgoshim University Grdute Shool of Medil nd Dentl Sienes, Skurgok, Kgoshim , Jpn e-mil: mskun@m.kufm.kgoshim-u..jp A. C. N. Mrhinti Fulty of Mediine, Jember University, Jember, Indonesi E. Arimur Mjor in Food nd Nutrition, Deprtment of Life nd Environmentl Siene, Kgoshim Prefeturl College, Kgoshim, Jpn thn PSe-db nd d-db mie. Deresed hepti triglyerides in PEx-db mie orresponded to lower expression of lipogeni enzyme genes in the liver. Moreover, PEx-db mie showed signifintly lower plsm brnhed-hin mino ids (BCAA), rginine, proline, nd tyrosine, in ddition to inresed skeletl musle (SM) weight, thn PSe-db nd d-db mie, in spite of little influene on the expression of the BCAA trnsminse gene, in SM nd WAT. Conlusion We found tht exerise under food restrition ondition dereses severl mino ids, inluding BCAA, nd my improve insulin sensitivity more thn mere food restrition. We propose tht the deresed onentrtion of blood mino ids my be vluble mrker evluting the effets of exerise on dibeti onditions. Keywords Animl model Exerise Gene expression Nutrition Type 2 dibetes mellitus Abbrevitions AA Amino id d Ad libitum BCAA Brnhed-hin mino ids PEx Pir-fed nd exerising FFA Free ftty ids HDL High-density lipoprotein PSe Pir-fed nd SLC Solute-linked rrier SM Skeletl musle T2DM Type 2 dibetes mellitus TG Triglyeride VE Voluntry exerise WAT White dipose tissue WT Wild type

2 34 Environ Helth Prev Med (214) 19: Introdution The inidene of type 2 dibetes mellitus (T2DM) hs reently grown worldwide [1, 2]. Although T2DM hs geneti predispositions, it is unlikely tht the gene pool hs hnged ppreibly over this short period of time [3, 4]. Thus, the growing inidene of T2DM my be relted to lifestyle ftors rther thn geneti ftors [1 4]. Exerise, one of these lifestyle ftors, hs mrkedly redued in the pst severl dedes, espeilly in developed ountries [1, 5, 6]. As pproprite exerise, the Centers for Disese Control nd Prevention nd the Amerin College of Sports Mediine hve jointly published reommendtions tht dults should engge in t lest 3 min of moderte physil tivity (e.g., brisk wlking) on most, nd preferbly ll, dys of the week [6]; therefore, the impt of exerise (long-term, moderte physil tivity, erobi, nd voluntry), representing hbitul exerise, on inresed gluose uptke nd ft oxidtion hs been eluidted [7, 8]. However, the effet of exerise on orgns suh s the liver nd brin, whih re diffiult to obtin, is diffiult to exmine in humns. Therefore, we re obliged to perform experiments using experimentl nimls t present. Using mouse models of T2DM, the effet of exerise on pthologil onditions, inluding gluose nd lipid metbolism hs been exmined [9 11]. In previous studies, voluntry exerise (VE) nd fored exerise, suh s tredmill nd swimming, hve been pplied [9 11]. VE my be more physiologil; however, mie with T2DM hve been reported to perform smll mount of exerise voluntrily [12, 13], whih is similr to people with T2DM [14]. In these reports, even VE filed to reverse defets in gluose nd insulin levels in db mie with leptin reeptor defiieny, n niml model of T2DM [12, 13]. On the other hnd, food restrition, pir-fed proedure suppressed weight gin, but did not restore ft ontent nd gluose levels [15, 16]. Reently, Strnhn et l. reported tht 6 % food restrition inresed VE in db mie, showing the effet of ombintion of food nd exerise interventions [17]. Strnhn et l. [17] reported the effet of exerise on metbolite levels of gluose nd lipid metbolism, nd brin funtion; however, the effet of exerise on gene expressions relted to gluose nd lipid metbolism hs not been exmined. Additionlly, the effet of exerise on mino id (AA) metbolism hs not been exmined. In nimls nd humns with type 2 DM, higher onentrtion of blood brnhed-hin mino ids hs been noted [18 2]; therefore, we exmined the effet of exerise on AA metbolism, in ddition to gluose nd lipid metbolism. The purpose of the present study ws to estblish n niml model of T2DM to evlute the effet of exerise in n niml model of T2DM under food restrition ondition by mens of pir-fed proedure. Using this model, we exmined the effets of exerise on AA metbolism, in ddition to gluose nd lipid metbolism in db mie. Additionlly, we tried to exmine the effets of exerise on mrna levels of the genes relted to nutrient metbolism in the liver, skeletl musle (SM), nd white dipose tissue (WAT), representing insulin-sensitive orgns. Mterils nd methods Animls nd pir-fed onditions Mle mie with dibetes [C57BLKS(BKS).Cg-? Lepr db /? Lepr db /J; db], hving homozygous muttion of the leptin reeptor, nd ontrol (BKS.Cg-Dok7 m?/ Dok7 m?/j; WT, mie without dibetes) mie t 4 weeks of ge were purhsed from Chrles River Jpn (Kngw, Jpn) [21]. After 3 dys limtion, s exerising (Ex) or (Se) onditions, mie were individully housed in ge with running wheel or loked wheel, respetively. They were kept t onstnt room temperture of 22 ± 2 C, with 12 h of illumintion (7: 19:). WT mie nd db mie were subjeted to the following 5 onditions for 25 dys: (1) d-wt mie: d libitum nd, (2) PEx-WT mie: pir-fed nd exerise, (3) d-db mie: d libitum nd, (4) PSe-db mie: pir-fed nd, nd (5) PEx-db mie: pir-fed nd exerise. Food (14.4 kj/g, CE-2; CLEA, Tokyo, Jpn), whih ws equl to the food onsumed by d-wt mie under the d libitum-fed ondition in 1 dy, ws supplied to the mouse group under pir-fed onditions every dy t 17: (food onsumption during the experimentl period: d-db mie: ± 7.3 g, pir-fed group mie: 13.5 g). On the lst dy for d-wt mie, we mesured the mount of food onsumed by the mie during 17: 19:, resulting in.8 g food per mouse. The next dy, we supplied.8 g food to the remining groups of mie t 17:, nd then killed the mie fter 2 h, mening tht the mie were treted s being under feeding onditions. Finlly, for nlysis, blood ws tken from the hert, nd then the orgns were removed. Blood prmeters nd mrna levels of the gene relted to AA, gluose, nd lipid metbolism in the liver, SM (soleus) nd WAT (ft surrounding epididymis) were exmined. The present study ws pproved by the Ethis Committee for Animl Experimenttion t Kgoshim University, whih is stndrdized to the Jpnese ntionl guideline for niml experiments, nd the priniples of lbortory niml re were followed.

3 Environ Helth Prev Med (214) 19: Biohemil prmeters Blood (.5 ml) ws drwn from the hert with syringe ontining.2 mol/l EDTA (1 ll), nd orgns inluding the hert, liver, SM, nd WAT were isolted [22]. The orgns were rinsed in isotoni sline, weighed, nd quikly frozen in liquid nitrogen. The blood ws entrifuged t 3, rpm for 5 min for seprtion of plsm. The plsm nd frozen orgns were stored t -8 C until nlysis. Leptin, insulin, diponetin, nd ortiosterone were mesured by the respetive ELISA kits (R&D Systems, Minnepolis, MN; Moring Institute of Biologil Siene In., Kngw, Jpn; Otsuk Phrmeutil Co., Tokyo, Jpn; Enzo Life Sienes In., Plymouth Meeting, PA, respetively). HbA1 ws mesured by n immunossy (DCA 2 system; Byer Dignostis, Elkhrt, IN). Plsm mino id onentrtions were determined with JLC-5 model mino id nlyzer (JEOL, Tokyo, Jpn) fter deproteiniztion with 5 % (w/v) sulfosliyli id [23]. Gluose, free ftty ids (FFA), triglyeride (TG), totl holesterol, nd highdensity lipoprotein (HDL) holesterol were mesured using the respetive kits (Wko, Osk, Jpn). The frozen liver ws weighed nd dded to lipid extrtion solution omposed of methnol hloroform, nd then homogenized. The orgni phse ws trnsferred to glss tubes, evported with nitrogen gs, reonstituted in n isopropnol-triton X-1 mixture (1 % v/v), nd then vortexed [24]. Lipid ontent ws mesured with kits (TG, TG-E test Wko; T-Cho, Cho- CII test Wko). Gene expressions The liver, SM, nd WAT were used for isoltion of totl RNA [25]. Isolted RNA ws treted with DNse tretment to remove genomi ontmintion. First-strnd DNA synthesis ws performed using 5 lg totl RNA with oligo- (dt) 2 primer following the mnufturer s instrutions (Invitrogen, Crlsbd, CA). Rel-time quntittive PCR ws performed using SYBR-green on TAKARA detetion system (TAKARA, Shig, Jpn) under the onditions reommended by the mnufturer. The primry sequenes of the genes re shown in supplementry Tble 1 [26, 27]. The reltive level of mrna ws lulted using yle time (Ct) vlues, whih were normlized ginst the vlue of b- tubulin. Reltive quntifition (fold hnge) between different smples ws then determined ording to the 2-44Ct method. Due to the high vrition in gene expression dt of d-wt, the level of the genes in d-db mie ws set t 1.. Sttistil nlyses Vlues re presented s the men ± stndrd error (SE). The unpired Student s t test ws pplied s pproprite to identify signifint differenes between groups, using Ekuseru-Tokei 28 softwre (SSRI Co., Ltd., Tokyo, Jpn), with P \.5 s the riterion. Results Running distne nd food intke We preliminrily mesured the mount of exerise by db mie t 4 weeks of ge under d libitum onditions. db Mie performed signifintly less exerise thn WT mie, represented s the wheel-running distne (.7 ±.1 vs. 4.5 ±.5 km/dy, N = 4; P \.5). Regrding food intke, d libitum db mie onsumed signifintly more food thn WT mie for the first week fter strting exerise (5.8 ±.1 vs. 4. ±.1 g/dy, N = 4; P \.5). Under pir-fed onditions where the mount of food onsumed by d-wt mie ws supplied to db mie, the db mie under the food restrition (Fig. 1A) performed more exerise thn d-db mie t 4 weeks (1.7 ± 1.3 vs..7 ±.1 km/dy, N = 6 nd N = 4, respetively). During the experimentl period for 4 weeks, WT mie performed n inresed mount of exerise under the onditions, wheres db mie showed lrgely onstnt vlues even under food restrition onditions (Fig. 1A). db Mie performed signifintly less exerise thn WT mie, exept 1 week fter strting the exerise. Body weight nd orgn weight As shown in Fig. 1B, ll groups of db mie weighed signifintly more thn WT mie t 4 weeks of ge before exerise. In 1 week, db mie under pir-fed ondition with nd without exerise weighed signifintly less thn d-db mie. Two weeks fter exerise under pir-feeding onditions, PEx mie with both geneti trits weighed signifintly less thn the mie under the ondition (d- WT nd PSe-db mie), respetively. Body weight gin is shown in Fig. 1C. PEx mie with both geneti trits hd signifintly lower body weight gin thn mie under the ondition (d-wt nd PSe-db mie), respetively. This my be due to inresed energy expenditure by exerise. As shown in Tble 1, PEx-db hd signifintly higher weight of SM, nd there ws tendeny towrd inresed weight of SM in PEx-WT (P =.13) s ompred to Se mie (d-wt nd PSe-db mie) with the respetive geneti trit. d-db Mie hd signifintly higher liver nd WAT weight thn the other 4 groups of mie (Tble 1). PSe- nd PEx-db mie showed signifintly lower hert weight thn d-db mie. PEx-db mie hd signifintly lower liver nd WAT weight thn PSe-db mie, s well s WT mie (Tble 1). Bsed on the rtio of

4 342 Environ Helth Prev Med (214) 19: A Running distne (km/dy) B Body weight (g) C Body weight gin (g) Age (weeks) Age (weeks) 5,, WT b,,,b, b, orgn weights divided by the body weight, PEx-WT mie showed signifintly lower rtio of WAT ompred to d- WT mie, nd signifintly higher rtio of SM for both geneti trits of mie ompred with mie under onditions., db,,b, b, b,,,b, b, Fig. 1 Body weight nd voluntry exerise during the experimentl period. Amount of voluntry exerise (A), body weight (B), nd body weight gin before exerise nd t 8 weeks (C) re shown. Cirles nd squres represent WT nd db mie, respetively. Closed symbols represent exerising mie under the pir-fed ondition (food mount onsumed by d libitum-fed WT mie ws supplied). Open symbols represent d libitum-fed mie under the ondition. Hthed symbols represent pir-fed db mie under the ondition (PSe-db mie). The vlues re the men ± SE (A), the mens - SE (B), or the mens? SE (), s pproprite. Vlues were nlyzed by Student s t test. In (A) nd (B), P \.5 vs. WT mie in eh physil ondition t the sme time point. b P \.5 vs. d-wt mie or PSe-db mie t the sme time point. P \.5 vs. d-db mie t the sme time point. In (C), P \.5 vs. WT mie in eh physil ondition. b P \.5 vs. d-wt mie or PSe-db mie. P \.5 vs. d-db mie Biohemil prmeters inluding hormones of gluose nd lipid metbolism As shown in Tble 1, PEx-db mie showed signifintly lower plsm insulin, leptin, nd ortiosterone, nd higher plsm diponetin thn PSe-db mie. For plsm gluose, PEx-db mie showed similr level to PSe-db mie, but signifintly lower level thn d-db mie. Regrding the reltionship between plsm gluose nd insulin, PEx-db mie showed similr result to d- nd PEx-WT mie, rther thn d- nd PSe-db mie, s shown in Fig. 2. This finding suggests tht exerise improved insulin sensitivity in db mie, orresponding to the results of HbA1 (Tble 1). Although PSe-db mie showed signifintly higher plsm ortiosterone thn d-db mie, the level in PEx-db mie ws signifintly lower thn in PSe-db mie. In the liver, PEx-db mie showed signifintly lower triglyeride ontent s mg/liver thn PSe-db mie. On the other hnd, PEx-db showed similr holesterol level to PSe-db mie (P =.43). Amino ids As shown in Tble 2, PEx-db mie showed signifintly lower rginine, isoleuine, leuine, ornithine, proline, tyrosine, nd vline thn PSe-db mie. As ompred to ddb mie, PSe-db nd PEx-db mie showed signifintly lower ystine, histidine, isoleuine, leuine, phenyllnine, nd tryptophn. Nmely, rginine, ornithine, proline, tyrosine nd vline were signifintly lower in PEx-db mie, but not in PSe-db mie thn in d-db mie. For BCAA (isoleuine, leuine nd vline), PEx-db mie showed signifintly lower level thn PSe-db nd d-db mie. Gene expressions relted to gluose, lipid, nd AA metbolism To revel the mehnism involved in the improvement of pthologil prmeters by VE, we exmined the mrna levels of the genes relted to gluose, lipid, nd AA metbolism. Ex-db mie showed signifintly lower expression of gluose trnsporter (Glut) 2 nd ftty id synthse (FAS) in the liver thn d-db mie (Fig. 3). There ws tendeny towrd lower etyl-coa rboxylse (ACC) 1 expression levels in the liver of PEx-db mie s ompred to d-db mie (P =.12). For gluose 6 phosphtse (G6Pse) expression, PEx-db mie showed similr levels to d-db mie, but there ws signifintly lower level in PSe-db mie s ompred to d-db mie. In phosphoenol pyruvte rboxykinse (PEPCK) expression, PEx-db mie showed similr levels to d-db nd PSe-db mie. In SM nd WAT, PEx-db mie showed signifintly

5 Environ Helth Prev Med (214) 19: Tble 1 Effet of voluntry exerise on orgn weight nd biohemil prmeters Group Fed ondition Physil ondition d-wt PEx-WT d-db PSe-db PEx-db d libitum (N = 4) Pir-fed exerising d libitum (N = 4) Pir-fed Pir-fed exerising Orgns Liver g 1.6 ±.2.98 ±.2 b, 2.92 ± ±.6, 1.26 ±.3,b, mg/g BW 46.7 ± ± ± ± ±.9, WAT g.29 ±.1.19 ±.1 b, 2.3 ± ±.6, 1.12 ±.6,b, mg/g BW 12.9 ±.4 9. ±.7 b, 47. ± ± ± 1.2, Hert mg 111 ± 2 19 ± ± 1 95 ± 2, 93 ± 4 mg/g BW 4.9 ± ± ± ±.1, 3.2 ±.1, SM (soleus) mg 7.1 ± ± ± ± ±.6 b mg/g BW.3 ±.1.5 ±. b,.2 ±..3 ±..4 ±.,b, Plsm Gluose mmol/l 1.4 ± ±.4 b, 34. ± ± ±.8 FFA mmol/l.4 ±.1.2 ±..5 ±.1.4 ±..4 ±.1 TG mmol/l.6 ±.1.6 ±.1 1. ±.1.7 ±.1.6 ±. Totl holesterol mmol/l 2.4 ± ±.2 4. ± ±.1, 2.3 ±.2 HDL-holesterol mmol/l 1.4 ± ±. b, 2.3 ± ±. 1.3 ±.1 Insulin pmol/l 167. ± ± ± ± ± 62.7,b, Leptin lg/l 6.8 ± ± ± ± ± 4.5,b, Adiponetin lg/ml 12.2 ± ±.9 b 13.2 ± ± ±.3 b, Cortiosterone nmol/l ± ± ± ± 42.8, 93.5 ± 63.2 b Blood HbA1 % 3.4 ± ± ± ±. 3.1 ±.1,b, (mmol/mol) (13.1 ± 1.3) (13.7 ±.7) (29.5 ± 1.6) (12.8 ±.3) (1.4 ±.8) Liver TG mg/g 3.7 ± ± ± ± 1.9, 9.9 ±.8, mg/liver 3.9 ±.4 3. ± ± ± 2.9, 12.6 ± 1.2,b, Cholesterol mg/g 1.4 ± ± ± ± ±.1, mg/liver 1.5 ± ±.1 b, 8.6 ± ±.2, 2.1 ±.1, Vlues re the men ± SE Vlues were nlyzed by Student s t test d d libitum-fed nd, PSe pir-fed nd, PEx pir-fed nd exerising, Pir-fed ondition food mount onsumed by d-wt mie ws supplied, BW Body weight, WAT white dipose tissue, FFA free ftty ids, TG triglyeride P \.5 vs. WT mie in eh physil ondition b P \.5 vs. d-wt or PSe-db mie P \.5 vs. d-db mie higher Glut4 nd FAS thn d-db mie. In ddition, ACC2 ws signifintly higher in SM of PEx-db mie, thn d-db mie. There ws tendeny towrd higher ACC1 expression levels in WAT of PEx-db mie, s ompred to d-db mie (P =.13). Regrding BCAA metbolism, the three groups of db mie showed similr expression levels in brnhed-hin minotrnsferse (BCAT) 2, solute-linked rrier (SLC) 3A2 nd 7A5 involved in BCAA trnsport, in SM nd WAT, respetively. Disussion In the present study, VE under pir-fed onditions improved gluose, lipid, nd AA metbolism in db mie more thn food restrition only, bsed on the results regrding blood HbA1, hepti TG ontent, nd blood BCAA levels. Moreover, the exerise differently ffeted expressions of the genes relted to lipid synthesis in insulin-sensitive orgns suh s the liver, SM, nd WAT.

6 344 Environ Helth Prev Med (214) 19: Gluose (mmol/l) b, Insulin (pmol/l) Fig. 2 Reltionship between plsm insulin nd gluose levels. Cirles nd squres represent WT nd db mie, respetively. Open symbols represent d libitum-fed mie under the ondition. Hthed symbols represent pir-fed db mie under the ondition. Closed symbols represent exerising mie under the pirfed ondition (food mount onsumed by d libitum-fed WT mie ws supplied). The vlues re the men ± SE. Vlues were nlyzed by Student s t test. For omprison of gluose nd insulin between the groups, lphbetil nd Greek letters re used for sttistil signifine, respetively. For gluose onentrtion; P \.5 vs. WT mie in eh physil ondition. b P \.5 vs. d-wt or PSe-db mie. P \.5 vs. d-db mie. For insulin onentrtion; P \.5 vs. WT mie in eh physil ondition. b P \.5 vs. d-wt or PSedb mie. P \.5 vs. d-db mie To the best of our knowledge, this is the first report to show suessful tretment without ny drugs for db mie, n niml model of T2DM. VE under pir-fed onditions redued body, liver, nd WAT weight in db mie. In ddition, body weight per se nd body weight gin fell in exerising mie (Fig. 1B, C), in ontrst to high SM weight. The inrese of the SM weight in db mie n be ounted for by n inrese in the SM weight itself, nd not only by derese in the body weight orresponding to higher rtio of the SM weight/body weight (Fig. 1B; Tble 1). Thus, the inresed SM weight despite body weight derese my be benefiil effet of exerise under the dibeti ondition, s ompred with mere food restrition. The deresed liver weight my be used by deresed TG ontent, suggesting tht exerise is effetive to suppress the development of ftty liver found in onditions with dibetes. This finding is onsistent with reports on humn studies [28, 29]. Interestingly, in the gene expression study, lipogeni enzyme genes suh s FAS nd ACC1 in liver were lower in PEx-db mie thn in d-db mie (Fig. 3). On the other hnd, in SM nd WAT, the lipogeni enzyme genes were rther inresed in PEx-db mie, inditing tht exerise my hve different influenes on lipid metbolism in the respetive orgns sensitive to insulin tion. Expression of Glut4 ws inresed in SM nd WAT of PEx-db mie. Moreover, during the present study, HbA1 ws shown to be lower in PEx-db mie. As shown in Fig. 2, exerise my enhne insulin sensitivity, rther thn only food restrition. Altogether, exess blood gluose in onditions with dibetes my be inorported into SM nd WAT bsed on high expression of GluT4 nd lipogeni enzyme genes, leding to lipid synthesis enhned by exerise s replenishment of triglyeride. This finding is onsistent with reports on humn studies [7, 8]. In rodents nd humns with dibetes onditions inluding type 1 nd type 2, inresed BCAA hs been reported [18 2]. The underlying mehnism is involved in insulin resistne [3, 31]. Very reently, inresed BCAA hs been reported to be n importnt prospetive ftor for the onset of T2DM [2]. In the present study, exerise deresed BCAA in db mie more thn food restrition in db mie. In db mie, brnhed-hin -keto ids suh s isovleri id showed lower exretion in urine [32], inditing the slow degrdtion nd trnsport of BCAA in WAT nd SM, whih re thought to ply role in BCAA metbolism [26, 27, 33]. The gene expressions of BCAT2, SLC3A2, nd SLC7A5, the responsible genes in BCAA degrdtion nd trnsport, were not ltered under exerise onditions in our models, exept BCAT2 in SM (Fig. 3). Although the detiled underlying mehnism hs not been lrified, VE ould derese blood BCAA to the WT mouse level, possibly due to improved insulin resistne (Fig. 2) nd/or inresed inorportion into skeletl musle, orresponding to enlrged skeletl musle relted to enhnement of protein synthesis (Tble 1). The enlrged skeletl musle by VE ws onsistent with the previous report [23]. In exerising db mie, the derese in rginine nd ornithine through possible inorportion into the liver, intermedites of the ure yle, suggests n importnt role in mmoni detoxifition [34]. Proline nd lnine (vs. Se-db mie, P =.6), whih derese with exerise, my be utilized effiiently to form gluose in the liver [35]. However, it remins to be eluidted whether exerise enhned hepti gluoneogenesis. Regrding the effet of exerise on hormone levels relted to nutrient metbolism, exerise inresed diponetin nd deresed leptin in blood (Tble 1), leding to enhned insulin sensitivity, s in humns [36, 37]. The ltered levels of blood diponetin nd leptin nnot be explined by gene expressions in WAT beuse exerise did not signifintly influene the expression level of diponetin nd leptin in WAT (dt not shown). The underlying mehnism regrding the influene of exerise on blood diponetin nd leptin levels will be eluidted by this niml model. Regrding ltertions of plsm ortiosterone levels, fsting hs been reported to inrese plsm ortiosterone [38, 39]. Interestingly, VE in the present study deresed plsm ortiosterone in db mie, suggesting tht the effets of exerise re not explined by the effets of longer fsting in ssoition with inresed energy expenditure.

7 Environ Helth Prev Med (214) 19: Tble 2 Plsm mino id onentrtions in WT nd db mie Group Fed ondition Physil ondition d-wt PEx-WT d-db PSe-db PEx-db d libitum (N = 4) Pir-fed exerising d libitum (N = 4) Pir-fed Pir-fed exerising Alnine 2. ± ± ± ± ± 2.7 Arginine 57.1 ± ± ± ± ± 5.1,b, Asprgine 25.9 ± ± ± ± ± 2.5 Asprti id 14. ± ± ± ± ± 2.5 Citrulline 51.7 ± ± 4.7 b 93.7 ± ± ± 6.6 Cystine 51.8 ± ± ± ± 1.6, 38.4 ± 2.7, Glutmi id 19. ± ± ± ± ± 2.3 Glutmine ± ± ± ± ± 38.4 Glyine ± ± ± ± ± 17.2 Histidine 45.4 ± ± 4.3 b, 76.5 ± ± ± 5., Isoleuine 74.2 ± ± ± ± 1.2, 77.7 ± 6.2 b, Leuine 19.8 ± ± ± ± 17.3, ± 9.4 b, Lysine ± ± ± ± ± 18.7 Methionine 51.3 ± ± 6.8 b, 39.9 ± ± ± 4.5 Ornithine 49.1 ± ± ± ± ± 3.7,b Phenyllnine 56.2 ± ± ± ± ± 5.9, Proline 73. ± ± ± ± ± 7.5,b Serine 96.3 ± ± 9.6 b, 9.5 ± ± ± 7.7 Threonine ± ± 17.1 b, ± ± ± 14.2 Tryptophn 49.8 ± ± ± ± ± 3.9 Tyrosine 61.9 ± ± ± ± ± 3.4,b, Vline ± ± ± ± ± 24.6 b, BCAA ± ± ± ± ± 39.4 b, Vlues re the men ± SE Vlues were nlyzed by Student s t test d d libitum fed nd, PSe pir-fed nd. PEx pir-fed nd exerising, Pir-fed ondition food mount onsumed by d-wt mie ws supplied, BCAA brnhed-hin mino ids P \.5 vs. WT mie in eh physil ondition b P \.5 vs. d-wt or PSe-db mie P \.5 vs. d-db mie To evlute the effet of exerise, VE should be ombined with food restrition. With food restrition, db mie performed reltively high mount of exerise ompred to d-db mie. This result is onsistent with report by Strnhn et l. [17]. The pir-fed ondition in the present study, one type of food restrition, my relieve the suppression of orexin-dopmine signling, leding to n inresed mount of VE, by improving hyperglyemi. Keller et l. [4] hve lredy shown tht the mount of VE performed orreltes with blood gluose in nimls with dibetes. The findings suggest tht dietry intervention reduing energy intke my inrese the motivtion to exerise, resulting in effiient effets of exerise on dibeti onditions. There is limittion of the study. The experiments were performed using n niml model with genetilly defiient leptin reeptor. Although the mie re n niml model of T2DM, they re not the sme s dibetes in humn ses. Although high blood leptin levels orresponding to leptin resistne re similr to the pthologil sitution in humn ses of T2DM [41], the results obtined here should be pplied utiously to humns. In onlusion, we hve estblished n niml model of T2DM evluting the effets of exerise on dibeti onditions by mens of pir-fed proedure. Using this model, we found tht exerise dereses severl mino ids, inluding BCAA, nd my improve insulin sensitivity more thn mere food restrition. In other words, the

8 346 Environ Helth Prev Med (214) 19: A 4 Referenes Fold hnge B Fold hnge C Fold hnge deresed AA onentrtion in blood my be utilized s vluble mrker to evlute the effiieny of exerise on dibeti onditions. This view will be evluted in further experiments in humns. Aknowledgments This study ws supported by grnt from the Ministry of Edution, Siene, Sports nd Culture of Jpn. We thnk Ms. Chiko Nishinosono for seretril ssistne nd Mr. Dniel Mrozek for opyediting. We wish to thnk the Joint Reserh Lbortory, Kgoshim University Grdute Shool of Mentl nd Dentl Sienes, for the use of their filities. We thnk ll the stff members of the Institute of Lbortory Animl Sienes, Kgoshim University who kept the nimls in good ondition. Conflit of interest * * Glut2 G6Pse PEPCK FAS ACC1 ** ** * * Glut4 FAS ACC2 BCAT2 SLC3A2 SLC7A5 *, # * ** Glut4 FAS ACC1 BCAT2 SLC3A2 SLC7A5 Fig. 3 Gene expressions in the liver (), skeletl musle (SM, b), nd white dipose tissue (WAT, ). The vlues re shown s reltive fold hnge over d libitum db (d-db) mie (ll vlues were set t 1.). Open olumn, d-db mie; hthed olumn, pir-fed nd db mie; losed olumn, pir-fed nd exerising db mie. Food mount onsumed by d libitum-fed WT mie ws supplied s the pir-fed ondition. The vlues re the mens? SE. Vlues were nlyzed by Student s t test. *P \.5, **P \.1 vs. d-db mie; # P \.5 vs. PSe-db mie None. * * 1. Zimmet P, Alberti KGMM, Shw J. Globl nd soietl implitions of the dibetes epidemi. Nture. 21;414(6865): Mgkos F, Ynnkouli M, Chn JL, Mntzoros CS. Mngement of the metboli syndrome nd type 2 dibetes through lifestyle modifition. Annu Rev Nutr. 29;29: Jfr-Mohmmdi B, MCrthy MI. Geneti of type 2 dibetes mellitus nd obesity- review. Ann Med. 28;4(1): Lindgren CM, MCrthy MI. Mehnism of disese: geneti insights into the etiology of type 2 dibetes nd obesity. Nt Clin Prt. 28;4(3): DiPietro L. Physil tivity in ging: hnges in ptterns nd their reltionship to helth nd funtion. J Gerontrol. 21;56A(2): Sigl RJ, Kenny GP, Wssermn DH, Cstned-Sepp C, White RD. Physil tivity/exerise nd type 2 dibetes. Dibetes Cre. 26;29(6): Del F, Lrsen JJ, Mikines KJ, Ploug T, Petersen LN, Glbo H. Insulin-stimulted musle gluose lerne in ptients with NIDDM. Effets of one-legged physil trining. Dibetes. 1995;44(9): Stllkneht B, Lrsen JJ, Mikines KJ, Simonsen L, Bülow J, Glbo H. Effet of trining on insulin sensitivity of gluose uptke nd lipolysis in humn dipose tissue. Am J Physiol Endorinol Metb. 2;279(2):E Tng T, Reed MJ. Exerise dds to metformin nd rbose effiy in db/db mie. Metbolism. 21;5(9): Hung H, Iid KT, Sone H, Yokoo T, Ymd N, Ajisk R. The effet of exerise trining on diponetin reeptor expression in KKAy obese/dibeti mie. J Endorinol. 26;189(3): Hskell-Luevno C, Shub JW, Andresen A, Hskell KR, Moore MC, Koerper LM, et l. Voluntry exerise prevents the obese nd dibeti metboli syndrome of the melnoortin-4 reeptor knokout mouse. FASEB J. 29;23(2): Sennott J, Morrissey J, Stndley PR, Broderik TL. Tredmill exerise trining fils to reverse defets in gluose, insulin nd musle GLUT4 ontent in the db/db mouse models of dibetes. Pthophysiology. 28;15(3): Prrot CR, Ghosh P, Tedeshi J, Gunsekr G, Broderik TL. Urinry ortiosterone nd normetnephrine levels fter voluntry wheel nd fored tredmill running in the db/db mouse. J Dibetes Mellitus. 211;1(4): Pn A, Lus M, Sun Q, vn Dm RM, Frno OH, Mnson JE, et l. Bidiretionl ssoition between depression nd type 2 dibetes mellitus in women. Arh Intern Med. 21;17(21): Cox JE, Powley TL. Development of obesity in dibeti mie pir-fed with len siblings. J Comp Physiol Psyhol. 1977;91(2): Ymmoto Y, Uet Y, Serino R, Nomur M, Shibuy I, Ymshit H. Effets of food restrition on the hypothlmi prepro-orexin gene expression in genetilly obese mie. Brin Res Bull. 2;51(6): Strnhn AM, Lee K, Mrtin B, Mrtin B, Mudsley S, Golden E, Cutler R, et l. Voluntry exerise nd lori restrition enhne hippompl dendriti spine density nd BDNF levels in dibeti mie. Hippompus. 29;19(1): Wijekoon EP, Skinner C, Brosnn ME, Brosnn JT. Amino id metbolism in the Zuker dibeti ftty rt: effets of insulin resistne nd of type 2 dibetes. Cn J Physiol Phrmol. 24;82(7): She P, Horn CV, Reid T, Hutson SM, Cooney RN, Lynh CJ. Obesity-relted elevtions in plsm leuine re ssoited with ltertion in enzymes involved in brnhed hin mino id

9 Environ Helth Prev Med (214) 19: metbolism. Am J Physiol Endorinol Metb. 27;293(6): E Wng TJ, Lrson MG, Vsn RS, Cheng S, Rhee EP, MCbe E, et l. Metbolite profiles nd the risk of developing dibetes. Nt Med. 211;17(4): Chen H, Chrlt O, Trtgli LA, Woolf EA, Weng X, Ellis SJ, et l. Evidene tht the dibetes gene enodes the leptin reeptor: identifition of muttion in the leptin reeptor gene in db/db mie. Cell. 1996;84(3): Arimur E, Horiuhi M, Kwguhi H, Miyoshi N, Aoym K, Tkeuhi T. Low-protein diet improves blood nd urinry gluose levels nd renl mnifesttions of dibetes in C57BLKS-db/ db mie. Eur J Nutr. 213;52(2): Tkeshit H, Horiuhi M, Izumo K, Kwguhi H, Arimur E, Aoym K, et l. Long-term voluntry exerise, representing hbitul exerise, lowers viserl ft nd lters plsm mino id levels in mie. Environ Helth Prev Med. 212;17(4): Wng Q, Li S, Jing L, Jing L, Zhou Y, Li Z, Sho M, et l. Defiieny in hepti ATP-itrte lyse ffets VLDL-triglyeride mobiliztion nd liver ftty id omposition in mie. J Lipid Res. 21;51(9): Chomzynski P, Shi N. Single-step method of RNA isoltion by id gunidinium thioynte-phenol-hloroform extrtion. Anl Biohem. 1987;162(1): She P, Reid TM, Bronson SK, Vry TC, Hjnl A, Lynh CJ, et l. Disruption of BCATm in mie leds to inresed energy expenditure ssoited with the tivtion of futile protein turnover yle. Cell Metb. 27;6(3): Drummond MJ, Fry CS, Glynn EL, Timmermn KL, Diknson JM, Wlker DK, et l. Skeletl musle mino id trnsporter expression is inresed in young nd older dults following resistne exerise. J Appl Physiol. 211;111(1): Be JC, Suh S, Prk SE, Rhee EJ, Prk CY, Oh KW, Prk SW, et l. Regulr exerise is ssoited with redution in the risk of NAFLD nd deresed liver enzymes in individuls with NAFLD independent of obesity in Koren dults. PLoS ONE. 212;7(1):e Rodriguez B, Torres DM, Hrrison SA. Physil tivity: n essentil omponent of lifestyle modifition in NAFLD. Nt Rev Gstroenterol Heptol. 212;12(6): Newgrd CB. Interply between lipids nd brnhed-hin mino ids in development of insulin resistne. Cell Metb. 212;15(5): Lu J, Xie G, Ji W, Ji W. Insulin resistne nd the metbolism of brnhed-hin mino ids. Front Med. 213;7(1): Knd ár R,Žáková P, Jirošová J, Sldká M. Determintion of brnhed hin mino ids, methionine, phenyllnine, tyrosine nd -keto ids in plsm nd dried blood smples using HPLC with fluoresene detetion. Clin Chem Lb Med. 29;47(5): She P, Zhou Y, Zhng Z, Griffin K, Gowd K, Lynh CJ. Disruption of BCAA metbolism in mie impirs exerise metbolism nd endurne. J Appl Physiol. 21;18(4): Krebs HA, Hems R, Lund P. Soures of mmoni for mmmlin ure synthesis. Biohem J. 1978;176(3): Krebs HA, Notton BM, Hems R. Gluoneogenesis in mouse-liver slies. Biohem J. 1966;11(3): Jürimäe J, Kums T, Jürimäe T. Plsm diponetin onentrtion is ssoited with the verge elerometer dily steps ounts in helthy elderly femles. Eur J Appl Physiol. 21;19(5): Shin-Efe A, Ktsikeris F, Mntzoros CS. Advnes in dipokines. Metbolism. 212;61(12): Mkimur H, Mizuno TM, Yng X-J, Silverstein J, Besley J, Mobbs CV. Cerulenin mimis effets of leptin on metboli rte, food intke, nd body weight independent of the melnoortin system, but unlike leptin, erulenin fils to blok neuroendorine effets of fsting. Dibetes. 21;5(4): Skoguhi T, Horiuhi M, Askw A, Ushiki M, Yoshid G, Fujimiy M, et l. Filure of the feeding response to fsting in rnitine-defiient juvenile viserl stetosis (JVS) mie: involvement of defetive yl-ghrelin seretion nd enhned ortiotropin-relesing ftor signling in the hypothlmus. Biohim Biophys At. 29;1792(11): Keller JB, Bevier WC, Jovnovi-Peterson L, Formby B, Durk EP, Peterson CM. Voluntry exerise improves glyemi in nonobese dibeti (NOD) mie. Dibetes Res Clin Prt. 1993;22(1): Mrtin SS, Qsim A, Reilly MP. Leptin resistne. J Am Coll Crdiol. 28;52(15):121 1.