Beneficial Effects of Aerobic Exercise Training Combined with Rosiglitazone on Glucose Metabolism in Otsuka Long Evans Tokushima Fatty Rats

Transcription

1 Originl Artile Oesity n Metoli Synrome Dietes Met J 217;41: pissn eissn DIABETES & METABOLISM JOURNAL Benefiil Effets of Aeroi Exerise Trining Comine with Rosiglitzone on Gluose Metolism in Otsuk Long Evns Tokushim Ftty Rts Shn-Ji Pio 1,2, *, So Hun Kim 1, *, Young Ju Suh 3, Seong-Bin Hong 1, Seong Hee Ahn 1, D He Seo 1, In-Sun Prk 4, Moonsuk Nm 1 1 Deprtment of Internl Meiine, Inh University Shool of Meiine, Inheon, Kore, 2 Qingo Enorine n Dietes Hospitl, Qingo, Chin, Deprtments of 3 Biomeil Sienes, 4 Antomy, Inh University Shool of Meiine, Inheon, Kore Bkgroun: Regulr eroi exerise is essentil for the prevention n mngement of type 2 ietes mellitus n my e prtiulrly enefiil for those trete with thizoliineiones, sine it my prevent ssoite weight gin. This stuy ime to evlute the effet of omine exerise n rosiglitzone tretment on oy omposition n gluose metolism in oese ietes-prone nimls. Methos: We nlyze metoli prmeters, oy omposition, n islet profiles in Otsuk Long Evns Tokushim Ftty rts fter 28 weeks of eroi exerise, rosiglitzone tretment, n omine exerise n rosiglitzone tretment. Results: Comine exerise with rosiglitzone showe signifintly less inrese in weight n epiiyml ft ompre to rosiglitzone tretment. Aeroi exerise lone n omine rosiglitzone n exerise tretment le to similr retention of len oy mss. All experimentl groups showe erese in fsting gluose. However, the omine exerise n rosiglitzone therpy group showe prominent improvement in gluose tolerne ompre to the other groups. Resue of islet estrution ws oserve in ll experimentl groups, ut ws most prominent in the omine therpy group. Conlusion: Regulr eroi exerise omine with rosiglitzone tretment n ompenste for the verse effet of rosiglitzone tretment n hs enefit for islet preservtion. Keywors: Bet-ell; Boy omposition; Dietes; Exerise; Rts, inre OLETF; Rosiglitzone INTRODUCTION Oesity is known to e mjor preisposing risk ftor for type 2 ietes mellitus (T2DM) [1]. Oesity my le to insulin resistne n β-ell ysfuntion [2,3]. Mjor strtegies for T2DM therpy fouses on the llevition of insulin resistne, improvement of β-ell funtion, n weight loss for oese ptients [4]. Rosiglitzone, thizoliineione (TZD) lss ntiieti gent, ts through tivting the nuler trnsription ftor peroxisome prolifertor-tivte reeptor γ [5-7]. In niml moels of insulin resistne, rosiglitzone hs een reporte to erese plsm gluose n insulin levels, improve insulin tion in skeletl musles, n prevent β-ell trophy [8]. It lso hs een reporte tht rosiglitzone promotes β-ell survivl n preserves β-ell mss in humn islet myloi polypeptie trnsgeni mie [8,9]. However, rosiglitzone Corresponing uthors: Moonsuk Nm Deprtment of Internl Meiine, Inh University Shool of Meiine, 27 Inhng-ro, Jung-gu, Inheon 22332, Kore E-mil: nmms@inh..kr In-Sun Prk Deprtment of Antomy, Inh University Shool of Meiine, 1 Inh-ro, Nm-gu, Inheon 22212, Kore E-mil: sunprk@inh..kr *Shn-Ji Pio n So Hun Kim ontriute eqully to this stuy s first uthors. Reeive: Mr. 2, 217; Aepte: Jul. 26, 217 This is n Open Aess rtile istriute uner the terms of the Cretive Commons Attriution Non-Commeril Liense ( whih permits unrestrite non-ommeril use, istriution, n reproution in ny meium, provie the originl work is properly ite. Copyright 217 Koren Dietes Assoition

2 Aeroi exerise omine with rosiglitzone uses weight gin s n verse effet, leing to overweight n oesity, whih my e mjor limittion of its use [1]. Aeroi exerise hs een tritionlly presrie for ietes prevention n mngement. Aeroi exerise hs enefiil effets on gluose metolism, minly through improvement of insulin sensitivity [11]. Aeroi exerise is n importnt omponent of lifestyle moifition interventions for weight loss. Therefore, eroi exerise tht n le to inrese energy onsumption my e enefiil in preventing the weight gin ssoite with the use of rosiglitzone [12]. Previous stuies hve shown the enefiil effets of omine exerise n rosiglitzone in improving riovsulr risk ftors in ptients with T2DM [13,14]. Benefiil effets of eroi exerise on β-ell preservtion hs een suggeste in niml n humn stuies [15]. However, the effet of omine regulr eroi exerise n rosiglitzone tretment on the pnres islet in T2DM hs not een evlute. We hypothesize tht the omintion of eroi exerise with rosiglitzone tretment n enhne positive effets y ompensting for the verse effet of weight gin use y TZDs, s well s y elerting the improvement of insulin resistne n β-ell preservtion. In the present stuy, we investigte hnges in oy omposition, gluose metolism, insulin resistne, n islet morphology in T2DM niml moel fter ministrting eroi exerise n rosiglitzone, lone n onomitntly. METHODS Animls The Otsuk Long Evns Tokushim Ftty (OLETF) rt is useful niml moel for T2DM with oesity [16]. At 5 to 6 weeks, the OLETF rt evelops oesity, while ieti symptoms pper t out 24 weeks [17]. Mle OLETF rts n ge-mthe mle Long-Evns Tokushim Otsuk (LETO) rts were supplie y Tokushim Reserh Institute, Otsuk Phrmeutils (Tokushim, Jpn). The rts were iniviully house in stnr niml fility with ess to stnr foo n wter liitum n mintine on 12-hour light-rk yle (lights on t 7: AM) in temperture- (22 C to 25 C) n humiity- (5% to 6%) ontrolle olony room. All proeures were in orne with institutionl guielines for niml reserh t Inh University in Inheon, the Repuli of Kore. Experimentl protool At 12 weeks of ge, totl of 56 OLETF rts were rnomly llote into four groups: (1) OLETF rts with no tretment (OC, n=14), (2) OLETF rts with rosiglitzone tretment only (OR, n=14), (3) OLETF rts with exerise only (OEx, n=14), n (4) OLETF rts with omine tretment of rosiglitzone n exerise (OREx, n=14). LETO rts were use s norml ontrols (LETO, n=14). Aitionl non-trete OLETF rts (n=7) n LETO rts (n=7) were srifie t seline (12 weeks ol) for pthologi exmintion. Rosiglitzone mlete (3 mg/kg/y, Avni; GSK In., Philelphi, PA, USA) ws orlly ministere to OR n OREx one y, 6 ys/week from week 12 s previously esrie [18] for 12 or 28 weeks. The OEx n OREx groups were sumitte to 3 minutes exerise t 15 m/min spee (low-intensity) on fore exerise wheel system (rt tivity ge; JD-A-6; Jungo B&P, Seoul, Kore) 5 ys week for 12 or 28 weeks. After 12 weeks (24 weeks ol) n 28 weeks (4 weeks ol) of tretment, intrperitonel gluose tolerne test (IPGTT) ws performe for the evlution of gluose tolerne, n the nimls were srifie for evlution of the pnreti islet ells n moleulr nlysis. Anlysis of loo smples Bloo smples were rwn fter n overnight fst, n the serum smples were kept t 8 C for susequent ssys. Insulin n leptin levels were etermine with enzyme-linke immunosorent ssy (ELISA) kits (Lino Reserh In., St. Chrles, MA, USA). Aiponetin levels were etermine with n ELI- SA kit (ALPCO Dignostis, Winhm, NH, USA). Gluose, totl holesterol, high ensity lipoprotein holesterol, low ensity lipoprotein holesterol, triglyeries, n free ftty is were ssesse with n utomte hemistry nlyzer (ADVIA 165; Byer, Tokyo, Jpn). A surrogte inex of insulin sensitivity ws lulte from fsting loo gluose n plsm insulin onentrtions s follows: homeostsis moel ssessment of insulin resistne (HOMA-IR)=(G I)/22.5, with gluose expresse s mmol/l n insulin expresse s μu/ml [19]. Gluose tolerne test The IPGTT ws performe s esrie previously [17]. Bloo gluose level ws nlyze using gluometer (One Touh Instrument; Rohe, Bsel, Swiss). Are uner the urve (AUC) for gluose ws lulte oring to the trpezoil rule from the gluose mesurements t seline, 3, 6, 9, 12, Dietes Met J 217;41:

3 Pio SJ, et l. n 18 minutes. Dul energy X-ry sorptiometry Boy omposition ws mesure t 3 weeks of ge y ulenergy X-ry sorptiometry (DEXA). Before unergoing whole-oy sn, the rts were nesthetize with Ketmine mixtures (Ketmine 35 mg/kg+xylzine 3 mg/kg+aetylpromzine.75 mg/kg), n then totl oy mss, one minerl ontent, len oy mss, n ft mss were mesure in vivo with QDR 45A DEXA using smll niml nlysis softwre (DEXA; Hologi In., Wlthm, MA, USA). The β-ell n islet morphology Immunoytohemistry ws performe s esrie previously [2]. For insulin stining, tissue setions were inute with nti-insulin ntioy (1:1,; Biogenex, Sn Rmon, CA, USA) for 24 hours t 4 C. In situ hyriiztion for insulin ws rrie out on tissue setions. After treting with proteinse K, the setions were inute with lkline phosphtse-onjugte nti-igoxigenin ntioy. Hyriiztion signls were revele y nitrolue tetrzolium n 5-romo-4-hloro-3-inolyl phosphte toluiinium. For the oule immunostining, tissue setions were trete with mouse nti-rt insulin ntioy (1:1,; Biogenex) n rit nti-rt gluose trnsporter 2 (GLUT-2) ntioy (1:1,; Alph Dignosti Interntionl, Sn Antonio, TX, USA). After pplying the seonry ntioy, fluoresein isothioynte (FITC) onjugte ntimouse n rhomine onjugte nti-rit ntioy (Jkson Immuno-Reserh L. In., West Grove, PA, USA) were e. The tissue setions were oserve using Rine 21 onfol mirosope (Bio-R n Nikon, Tokyo, Jpn). For morphometri nlysis, insulin-positive ells were ounte in the entire re of the tissue setions, n β-ell numer per unit re (1 μm 2 ) ws lulte s reporte previously [21]. To ssess islet preservtion, the islet estrution rtio (%) ws lulte s (the numer of estrute islets)/(the totl islet numer evlute) 1 (%). For eh pnres, two slies were reviewe t 1 mgnifition n 2 photos without overlp were rnomly tken to ount the numer of islets. The orresponing islets were exmine t 4 mgnifition to evlute whether there were signs of estrution. Destrute islets were efine s hving lymphoyte infiltrtion n istorte outline isling ler emrtion of the islet re. The men of the islet estrution rtios of the two slies ws lulte. Imge files were nlyze using Quntity One (Bio- R, Herules, CA, USA). Sttistil nlysis Nonprmetri tests were performe to ompre the metoli prmeters, oy weight, n oy omposition of the LETO, OC, OR, OEx, n OREx groups t eh ge point, ue to the reltively smll smple size of eh group. The Mnn- Tle 1. The hrteristis of the experimentl groups fter 28 weeks of tretment (t 4 weeks of ge) with rosiglitzone, exerise, n omine rosiglitzone n exerise Chrteristi LETO OC OR OEx OREx No Gluose, mmol/l 5.63 ( ) 7.27 ( ) 6.7 ( ) 6.24 ( ) 5.99 ( ) Insulin, ng/ml 3.24 ( ) 7.75 ( ) 2.65 ( ) 3.82 ( ) 2.43 ( ) Aiponetin, μg/ml 1.55 ( ) 1.5 ( ) 4.35 ( ) 1.72 ( ) 2.77 ( ) Leptin, ng/ml 2.91 ( ) ( ) ( ) 8.9 ( ), 1.66 ( ) Totl holesterol, mmol/l 2.59 ( ) 3.5 ( ) 2.52 ( ) 2.62 ( ) 2.4 ( ) HDL-C, mmol/l.76 (.74.78).91 (.82.95).82 (.76.86).82 (.76.93).81 (.78.85) Triglyeries, mmol/l.35 (.31.61) 1.63 ( ).42 (.34.76).74 (.51.81).51 (.48.58) Free ftty i, g/l.26 (.25.28).33 (.32.34).28 (.26.31).31 (.3.33).31 (.27.34) HOMA-IR ( ) 59.7 ( ) 17.7 ( ) ( ) 16.8 ( ) Vlues re presente s mein (interqurtile rnge). LETO, Long-Evns Tokushim Otsuk; OC, Otsuk Long Evns Tokushim Ftty (OLETF) rts with no tretment; OR, OLETF rts with rosiglitzone tretment only; OEx, OLETF rts with exerise only; OREx, OLETF rts with omine tretment of rosiglitzone n exerise; HDL- C, high ensity lipoprotein holesterol; HOMA-IR, homeostsis moel ssessment of insulin resistne. P<.5 vs. LETO, P<.5 vs. OC, P<.5 vs. OR. 476 Dietes Met J 217;41:

4 Aeroi exerise omine with rosiglitzone Whitney U test ws use to ompre the gluose tolerne levels n AUCs etween LETO n OC groups t the 12-week ge point. We ompre oy weight, oy omposition, gluose metoli prmeters, n AUCs mong LETO, OC, OR, OEx, n OREx groups t 24 n 4 weeks using the Kruskl- Wllis test n Tukey metho, using rnks for multiple omprisons. Bonferroni justments for multiple tests were itionlly rrie out to ompre oy n epiiyml ft weight vriles. SPSS version 14. (SPSS In., Chigo, IL, USA) ws use for sttistil nlysis. A level of 5% ws use for sttistil signifine. RESULTS Metoli hrteristis Tle 1 reports the metoli prmeters of the LETO n OLETF rts of eh group t 4 weeks (fter the 28-week intervention). The plsm gluose n insulin levels were higher in the OC group, ut reue in the OR, OEx, n OREx groups. Serum iponetin onentrtion ws elevte in the OR (P<.1) n OREx groups (P<.1). The serum leptin onentrtion ws erese in the OEx group (P<.1). Regring plsm lipi profiles, signifint ereses in serum totl holesterol (P<.5 n P<.1, respetively) n triglyerie (P=.2 n P=.3, respetively) levels were oserve in oth the OR n OREx groups. HOMA-IR, surrogte mrker of insulin resistne, ws signifintly inrese in the OC group ompre to the LETO group. The OR, OEx, n OREx groups ll showe improve insulin sensitivity ompre to the OC group to similr extent (Tle 1). Effets of rosiglitzone n exerise on gluose tolerne The IPGTT ws performe t 12, 24, n 4 weeks of ge to ssess gluose tolerne (Fig. 1). A greter inrese in loo gluose levels in response to the intrperitonel gluose lo ws seen in the OC group ompre to LETO (P<.1) (Fig. 2A- C). At 24 weeks, the OC group showe signifintly higher fsting gluose levels ompre to the LETO group, n the OREx group showe lower fsting gluose levels ompre to the OC group (Fig. 2B). At 4 weeks, fsting plsm gluose ws signifintly higher in the OC group ompre to LETO, n the OR, OEx, OREx groups ll showe lower fsting Fig. 1. Experimentl esign. Flow hrt of the experimentl esign is shown. LETO, Long-Evns Tokushim Otsuk; OLETF, Otsuk Long Evns Tokushim Ftty. Dietes Met J 217;41:

5 Pio SJ, et l Plsm gluose (mmol/l) Plsm gluose (mmol/l) LETO OC OR OEx OREx Time (min) A Time (min) B Plsm gluose (mmol/l) AUC (mmol/l min) 4, 3, 2, 1, LETO OC OR OEx OREx Time (min) C Age (wk) D Fig. 2. Effets of rosiglitzone n exerise tretments on gluose tolerne. Time ourse of loo gluose uring the intrperitonel gluose tolerne test is reporte t (A) 12 weeks, (B) 24 weeks, n (C) 4 weeks of ge. (D) The re uner the urve (AUC) for gluose t 4 weeks were lulte. Vlues re presente s men±stnr error (n=7). LETO, Long-Evns Tokushim Otsuk; OC, Otsuk Long Evns Tokushim Ftty (OLETF) rts with no tretment; OR, OLETF rts with rosiglitzone tretment only; OEx, OLETF rts with exerise only; OREx, OLETF rts with omine tretment of rosiglitzone n exerise. P<.5 vs. OC, P<.1 vs. OC, P<.5, P<.1 vs. LETO. plsm gluose level ompre to the OC group (Fig. 2C). Gluose tolerne ws further impire with ging for the OC group, ut signifint improvement in gluose tolerne ws seen in the OR, OEx, n OREx groups t 24 n 4 weeks. The OREx group showe further improvement ompre to the OR n OEx groups (Fig. 2B n C). At 24 weeks of ge, the gluose AUCs of the OREx group were signifintly lower thn those of the OC group, while no signifint hnges were foun in the OR n OEx groups. At 4 weeks, the OR, OEx, n OREx groups ll showe lower gluose AUCs ompre to the OC group, n the OREx group showe further improvement ompre to the OR n OEx groups (Fig. 2D). Effets of rosiglitzone n exerise on oy weight n omposition Chnges of oy weight n viserl ft re presente in Fig. 3. At 12 weeks of ge, OLETF rts emonstrte signifintly higher oy weight thn the LETO ontrols (393.8 [377.5 to 48.] vs. 32. [37.5 to ], P<.1) (Fig. 3A). At 4 weeks of ge (fter 28 weeks of tretment), the OR group emonstrte signifintly higher weight gin (1.78 [1.7 to 1.93] fol) ompre to the OC group (1.69 [1.64 to 1.76] fol), wheres weight gins were onsierly lower in the OEx (1.62 [1.52 to 1.7] fol) n OREx (1.57 [1.49 to 1.6] fol) groups (Fig. 3B). This shows tht rosiglitzone inue unesirle 478 Dietes Met J 217;41:

6 Aeroi exerise omine with rosiglitzone Boy weight (g) 6 4 LETO OC OR OEx OREx Chnge rtio Weeks 4 Weeks A LETO OC OR OEx OREx B 14 e 6 e Epiiyml ft weight (g) LETO OC OR OEx OREx Chnge rtio Weeks 4 Weeks C LETO OC OR OEx OREx D Fig. 3. Effets of rosiglitzone n exerise on oy weight n epiiyml ft weight. (A) Boy weight n (C) epiiyml ft weight t 12 n 4 weeks re shown. Vlues re presente s men±stnr error (n=7). The hnge rtio of (B) oy weight n (D) epiiyml ft weight from 12 to 4 weeks were lulte. Eh ox (n=7) re presente s mein (interqurtile rnge). LETO, Long-Evns Tokushim Otsuk; OC, Otsuk Long Evns Tokushim Ftty (OLETF) rts with no tretment; OR, OLETF rts with rosiglitzone tretment only; OEx, OLETF rts with exerise only; OREx, OLETF rts with omine tretment of rosiglitzone n exerise. P<.5 vs. LETO, P<.1 vs. LETO, P<.1 vs. OC, P<.1, e P<.1. Tle 2. Effets of rosiglitzone n exerise on oy omposition t 3 weeks of ge Vrile LETO OC OR OEx OREx No Totl mss, g ( ) ( ) ( ) ( ), ( ), Len oy mss, g ( ) ( ) ( ) ( ), 45.5 ( ), Ft mss, g 74. ( ) ( ) ( ), ( ), ( ), Vlues re presente s mein (interqurtile rnge). LETO, Long-Evns Tokushim Otsuk; OC, Otsuk Long Evns Tokushim Ftty (OLETF) rts with no tretment; OR, OLETF rts with rosiglitzone tretment only; OEx, OLETF rts with exerise only; OREx, OLETF rts with omine tretment of rosiglitzone n exerise. P<.5 vs. LETO, P<.5 vs. OR, P<.5 vs. OC. weight gin, n tht this weight gin n e resue y regulr eroi exerise. Epiiyml ft in the experimentl nimls prllele hnges in oy weight (Fig. 3C n D), implying tht weight ontrol y eroi exerise is ompnie Dietes Met J 217;41:

7 Pio SJ, et l. LETO OC OR OEx OREx 12 weeks 24 weeks 4 weeks A Destrution islet (%) β-cell numer/1 μm LETO OC OR OEx OREx B LETO OC OR OEx OREx C LETO OC OR OEx OREx D Fig. 4. Profiles of the pnreti islets in rosiglitzone, exerise, n omine tretment. (A) Chronologil hnges of pnreti islets in LETO rts n OLETF rts with n without nti-ieti tretment re presente with insulin immunostining t 12, 24, n 4 weeks. (B) Chnges in islet ltertions y perentge t 4 weeks. The numer of the ltere islets ws ounte in the pnreti tissues n the rtio ws lulte. Eh ox (n=7 2 setions) re presente s mein (interqurtile rnge). (C) β-cell numer of the totl re of the pnres ws lulte t 4 weeks. The pnreti islet re ws mesure using Quntity One (Bio-R). (D) In situ hyriiztion: insulin mrna in LETO, OC, OR, OEx, n OREx groups t 4 weeks. LETO, Long-Evns Tokushim Otsuk; OC, Otsuk Long Evns Tokushim Ftty (OLETF) rts with no tretment; OR, OLETF rts with rosiglitzone tretment only; OEx, OLETF rts with exerise only; OREx, OLETF rts with omine tretment of rosiglitzone n exerise. P<.1 vs. LETO, P<.1 vs. OC, P<.1 vs. OC, P<.1. y suppresse viserl ft inrese. These finings were losely relte to the oy omposition evlute y DEXA t 3 weeks of ge (Tle 2), showing signifint improvement in omposition of oth len oy mss n ft oy mss in the OEx n OREX groups ompre to the OR group. There ws no signifint ifferene in foo intke mong the groups, lthough there ws tren of inrese intke in the OC group ompre to the LETO group. Effets of rosiglitzone n exerise on islet morphology Fig. 3A shows hronologil hnges in islets in the LETO n OLETF rts. All experimentl rts retine norml islet fetures with roun or ovl shpe, ompt ellulr mss, n istint emrtion from the exorine tissue t 12 weeks of ge. Development of typil islets were etete t 24 weeks of ge in OLETF rts. The islets were somewht inflte n strte to lose outer emrtion, prtiulrly in the OC 48 Dietes Met J 217;41:

8 Aeroi exerise omine with rosiglitzone LETO OC OR OEx OREx Insulin GLUT2 Merge Fig. 5. Gluose trnsporter 2 (GLUT-2) expression in pnreti islets with rosiglitzone, exerise, n omine tretment t 4 weeks. The pnreti tissues were immunolele for insulin (green fluoresene in upper pnel) n GLUT-2 (re fluoresene in mile pnel). The immunofluoresent signls were merge n re shown in the lower pnel. LETO, Long-Evns Tokushim Otsuk; OC, Otsuk Long Evns Tokushim Ftty (OLETF) rts with no tretment; OR, OLETF rts with rosiglitzone tretment only; OEx, OLETF rts with exerise only; OREx, OLETF rts with omine tretment of rosiglitzone n exerise. group, wheres the islets of the LETO ontrol group emonstrte no ltertion t ll. Altertions of the islet were ggrvte t 4 weeks in the OC group, whih showe looser islet onstitution owing to inrese fier sustne s well s lymphoyte infiltrtion. The β-ells in the islets isply reltively wek immunoretivity for insulin (Fig. 4A). However, improvement in islet preservtion ws otine y rosiglitzone tretment s well s y regulr eroi exerises. The islets of the OR n OEx groups revele reltively well-orgnize islet fetures retining ompt β-ell mss with istinguishle ontours. Moreover, preservtion of the islet ws prominent in the OLETF rts trete with omine rosiglitzone n eroi exerise (OREx) whih showe ompt islet mss with strong insulin immunoretive ells (Fig. 4A). To ssess islet preservtion, we ssesse the islet estrution rtio s the perentge of islets showing n pprent morphologil ltertion (Fig. 4B). The β-ells mss ws estimte y ounting the numer of β-ells per unit re (μm 2 ) of the sme tissue setions use for the etermintion of ltere islets (Fig. 4B). Altertion n estrution of the islet ws foun to e severe in the OC group, espeilly t the ge of 4 weeks (>8%). However, the islets of the OR n OEx groups emonstrte signifint reution of this islet ltertion n estrution. Furthermore, we foun remrkle improvement of islet preservtion in experimentl rts trete with the omintion of rosiglitzone n eroi exerise (OREx) ompre to the OR n OE groups. Only smll numer of ltere islets (<5%) ws etete in the OREx group. We lso oserve signifint inrese of β-ell numers in the OR, OEx, n OREx groups t 4 weeks of ge (Fig. 4C). Insulin expression in β-ells ws onfirme t the insulin mrna level y in situ hyriiztion, s n inition of insulin synthesis (Fig. 4D). Expression signl of insulin mrna in the OC group ws lower ompre to the LETO ontrols, wheres the hyriiztion signls ppere to e improve in the OR, OEx n OREx groups. Pnreti GLUT-2 expression To estimte gluose sensitivity of the β-ells, we exmine GLUT-2 immunoretivity of the β-ell memrne (Fig. 5). We foun sustntil loss of GLUT-2 immunoleling in the β-ells of the OC rts ompre to the LETO ontrols, ut intt expression of GLUT-2 in the β-ells of the OR, OEx, n OREx rts. DISCUSSION Rosiglitzone is n ntiieti gent use in ptients with Dietes Met J 217;41:

9 Pio SJ, et l. T2DM to improve insulin sensitivity n gluose metolism [5,6,22]. Rosiglitzone hs superior urility when use for long perio of time ompre to other ntiieti gents, proly ue to its enefiil effet on preserving β-ell funtion [23]. However, the verse effet of oy weight gin [1] my limit its linil use. Therefore, n effort to limit unwnte weight gin will e neessry, n suffiient regulr eroi exerise my e enefiil in this linil setting [24]. In this stuy, we evlute the ntiieti effets of eroi exerise n its synergi vntges y omining it with TZD tretment. The omintion of eroi exerise n rosiglitzone tretment ws le to further improve gluose tolerne, fvorly ffet oy omposition, n prevent estrution of pnreti islets in OLETF rts s n itionl enefit. Severl stuies hve emonstrte tht suffiient mount of exerise n reue oy weight or prevent regin in oth humns n rts [11,22,24,25]. We foun tht regulr eroi exerise effetively mitigtes spontneous oesity s well s rosiglitzone-inue weight gin in OLETF rts. Exerise is lso of gret enefit sine it n reue ominl oesity, viserl ft, n riovsulr risk ftors, n inrese oth skeletl musle mss n riorespirtory fitness, even without signifint weight loss [26]. Inee, eroi exerise resulte in suessful ttenution of totl ft mss n viserl ft mss inreses use y rosiglitzone suggesting tht the mjor ostle of TZDs therpy n e eliminte y physil exerise. Exerise n rosiglitzone s omintion tretment ws shown to hve omplementry n itionl enefiil effets on glyemi inexes, insulin sensitivity, n riovsulr risk ftors ompre to either exerise or rosiglitzone y itself in previous stuies [13,14,27-3]. In ptients with T2DM, simultneous tretment with rosiglitzone n exerise ounterte rosiglitzone inue weight gin, extene improvements of insulin sensitivity, glyemi ontrol n fitness eyon those expete y their omplementry tions in ptients with T2DM fter 8 months [13]. In oese Zuker ftty rts, exerise n troglitzone lone eh h enefiil effet on insulin sensitivity, while the omintion of oth tretments ompletely normlize insulin sensitivity to the level of len ontrol rts. Exerise n troglitzone isplye ifferent mehnisms, n therefore the omine effet ws le to hve itionl enefits [28]. The present stuy shows tht t 4 weeks (fter 28 weeks of tretment) groups trete with exerise lone or rosiglitzone lone showe improve gluose tolerne, n the omine rosiglitzone n exerise tretment group showe more prominent improvement in gluose tolerne, in orne with previous stuies. Aiponetin ws signifintly inrese in the OR n OREx groups, while there ws no signifint hnge in the OEx group. This fining is onsistent with previous stuies in ptients with T2DM [13]. The inrese in plsm iponetin level y rosiglitzone tretment, whih is not seen in the exerise-lone group, woul e n itionl enefit of omining rosiglitzone to exerise, sine iponetin is known to hve enefiil effets suh s insulin sensitizing, nti-inflmmtory, n nti-therogeni properties [31]. Although we hve not mesure other ytokines, it is possile tht other ytokines my hve lso plye role. In ptients with T2DM, exerise, rosiglitzone, n omine tretment ll suppresse interleukin 6 (IL-6), resistin, n IL-18, n inrese IL-1 levels, while oth omine tretment n rosiglitzone tretment lone erese tumor nerosis ftor α levels signifintly [13,14]. In ition to the known enefiil effets of the omine tretment of eroi exerise n rosiglitzone, the urrent stuy shows the enefit of preservtion of pnres islet morphology in T2DM rt moel. This is the first stuy to evlute pnres morphology fter onurrent tretment with exerise n TZD. Sine iret evlution of the pnres fter tretment is not fesile in humn stuies, this niml t shoul e informtive in proviing knowlege on the effets of omine tretment in the pnres. Dietes is losely ssoite with efets of islet β-ells, n β-ell ysfuntion is n erly event leing to the evelopment of T2DM [32]. In oese humns, there is n inrese in β-ell mss ompre to nonoese humns. Oese ptients with impire fsting gluose n T2DM h signifintly erese β-ell mss ompre to non-ieti people [33]. This oinies with signifint ltertions of pnreti islets s shown in non-trete ieti OLETF rts. Both rosiglitzone [8,32,34] n exerise [15,35, 36] hve een reporte to hve enefiil effets on pnreti islet preservtion in nimls. The present stuy further shows tht eroi exerise protets islet estrution prtiulrly when omine with rosiglitzone tretment. Hyperglyemi, hyperlipiemi, n inflmmtion re thought to ontriute to β-ell ysfuntion [32]. Rosiglitzone hs een shown to improve inies of β-ell funtion when ssesse y the HOMA in ptients with T2DM. Exerise lso hs een shown to improve β-ell funtion mesure y the isposition inex [15,37]. The enefit of oth exerise n rosiglitzone on the pnres my e iniret, minly y reuing the seretory e- 482 Dietes Met J 217;41:

10 Aeroi exerise omine with rosiglitzone mn, ut there is eviene supporting iret enefiil effets on the pnres, espeilly for rosiglitzone [38,39]. In summry, the present stuy emonstrtes tht omine rosiglitzone with regulr eroi exerise n prominently improve gluose metolism, n this is ssoite with protetion from islet estrution in T2DM prone-niml moel. Comine regulr eroi exerise with rosiglitzone ws lso le to ttenute the inrese in oy weight n ft mss inue y rosiglitzone tretment. It is notiele tht eroi exerise n rosiglitzone n omplement eh other n le to itionl enefits when omine, n n le to etter sustine glyemi ontrol in orne with its itionl enefit on islet preservtion. Therefore, prtiipting in regulr eroi exerise shoul e strongly enourge to ptients with T2DM in onjuntion with TZD presription for etter therpeuti effets, n to prevent the sie effets of TZDs. CONFLICTS OF INTEREST No potentil onflit of interest relevnt to this rtile ws reporte. ACKNOWLEDGMENTS This stuy ws supporte y grnt of the Kore Helthre tehnology R&D Projet Ministry of Helth n Welfre, Repuli of Kore (HI14C162). Shn-Ji Pio reeive grnt supporte y the Brin Kore 21 Projet, Repuli of Kore. This work ws supporte y the KOSEF (26-542, R , n ) n NRF (217R1D- 1A1B334581), Repuli of Kore. REFERENCES 1. Kriketos AD, Crey DG, Jenkins AB, Chisholm DJ, Furler SM, Cmpell LV. Centrl ft preits eteriortion of insulin seretion inex n fsting glyemi: 6-yer follow-up of sujets t vrying risk of type 2 ietes mellitus. Diet Me 23;2: Khn SE. Clinil review 135: the importne of et-ell filure in the evelopment n progression of type 2 ietes. J Clin Enorinol Met 21;86: Gulstrn M, Ahren B, Amson U. Improve et-ell funtion fter stnrize weight reution in severely oese sujets. Am J Physiol Enorinol Met 23;284:E Inzuhi SE, Bergenstl RM, Buse JB, Dimnt M, Ferrnnini E, Nuk M, Peters AL, Tsps A, Wener R, Mtthews DR. Mngement of hyperglyemi in type 2 ietes: ptiententere pproh. Position sttement of the Amerin Dietes Assoition (ADA) n the Europen Assoition for the Stuy of Dietes (EASD). Dietologi 212;55: Ferre P. The iology of peroxisome prolifertor-tivte reeptors: reltionship with lipi metolism n insulin sensitivity. Dietes 24;53 Suppl 1:S Dn SL, Hoener PA, Bilkovis JM, Cromie DL, Ogilvie KM, Kuffmn RF, Mukherjee R, Pterniti JR Jr. Peroxisome prolifertor-tivte reeptor sutype-speifi regultion of hepti n peripherl gene expression in the Zuker ieti ftty rt. Metolism 21;5: Etgen GJ, Olhm BA, Johnson WT, Broerik CL, Montrose CR, Brozinik JT, Misener EA, Ben JS, Bensh WR, Brooks DA, Shuker AJ, Rito CJ, MCrthy JR, Areky RJ, Tyhons JS, Dn SL, Bilkovis JM, Pterniti JR Jr, Ogilvie KM, Liu S, Kuffmn RF. A tilore therpy for the metoli synrome: the ul peroxisome prolifertor-tivte reeptor-lph/ gmm gonist LY46568 meliortes insulin resistne n ieti hyperglyemi while improving riovsulr risk ftors in prelinil moels. Dietes 22;51: Koh EH, Kim MS, Prk JY, Kim HS, Youn JY, Prk HS, Youn JH, Lee KU. Peroxisome prolifertor-tivte reeptor (PPAR)- lph tivtion prevents ietes in OLETF rts: omprison with PPAR-gmm tivtion. Dietes 23; 52: Hull RL, Shen ZP, Wtts MR, Kom K, Crr DB, Utzshneier KM, Zrik S, Wng F, Khn SE. Long-term tretment with rosiglitzone n metformin reues the extent of, ut oes not prevent, islet myloi eposition in mie expressing the gene for humn islet myloi polypeptie. Dietes 25;54: Umrni DN, Bny AA, Hussin T, Lokhnwl MF. Rosiglitzone tretment restores renl opmine reeptor funtion in oese Zuker rts. Hypertension 22;4: Colerg SR, Sigl RJ, Fernhll B, Regensteiner JG, Blissmer BJ, Ruin RR, Chsn-Ter L, Alright AL, Brun B; Amerin College of Sports Meiine; Amerin Dietes Assoition. Exerise n type 2 ietes: the Amerin College of Sports Meiine n the Amerin Dietes Assoition. Joint position sttement. Dietes Cre 21;33:e Amerin Dietes Assoition. Stnrs of meil re in ietes: 215. Summry of revisions. Dietes Cre 215;38 Suppl:S4. Dietes Met J 217;41:

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12 Aeroi exerise omine with rosiglitzone 35. Clegri VC, Arntes JL, Silveir LR, Pul FM, Cost JM Jr, Rfho A, Velloso LA, Crneiro EM, Bosqueiro JR, Boshero AC, Zoppi CC. Enurne trining stimultes growth n survivl pthwys n the reox lne in rt pnreti islets. J Appl Physiol (1985) 212;112: Shim K, Shi K, Mizuno A, Sno T, Ishi K, Nom Y. Exerise trining hs long-lsting effet on prevention of non-insulinepenent ietes mellitus in Otsuk-Long-Evns-Tokushim Ftty rts. Metolism 1996;45: Slentz CA, Tnner CJ, Btemn LA, Durheim MT, Huffmn KM, Houmr JA, Krus WE. Effets of exerise trining intensity on pnreti et-ell funtion. Dietes Cre 29;32: Gupt D, Peshvri M, Mong N, Jetton TL, Lehy JL. Physiologi n phrmologi moultion of gluose-epenent insulinotropi polypeptie (GIP) reeptor expression in etells y peroxisome prolifertor-tivte reeptor (PPAR)- gmm signling: possile mehnism for the GIP resistne in type 2 ietes. Dietes 21;59: Meiute Arviiene S, Lunquist I, Glvnovskis J, Flogren E, Ole B, Slehi A. Plmitte-inue et-ell ysfuntion is ssoite with exessive NO proution n is reverse y thizoliineione-meite inhiition of GPR4 trnsution mehnisms. PLoS One 28;3:e Dietes Met J 217;41: