Supplemental epilactose prevents metabolic disorders through uncoupling protein-1 induction in the skeletal muscle of mice fed high-fat diets

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1 British Journl of Nutrition (215), 114, The Authors 215 doi:1.117/s Supplementl epiltose prevents metoli disorders through unoupling protein-1 indution in the skeletl musle of mie fed high-ft diets Yuki Murkmi 1, Teruyo Ojim-Kto 2, Wtru Suri 3, Hruhide Mori 3, Hirokzu Mtsui 3, Soihi Tne 1 nd Tkuy Suzuki 1 * 1 Deprtment of Biofuntionl Siene nd Tehnology, Grdute Shool of Biosphere Siene, Hiroshim University, Higshi-Hiroshim , Jpn 2 Deprtment of Bioengineering Sienes, Grdute Shool of Biogriulturl Sienes, Ngoy University, Ngoy , Jpn 3 Division of Applied Biosiene, Reserh Fulty of Agriulture, Hokkido University, Spporo , Jpn (Sumitted 21 My 215 Finl revision reeived 3 July 215 Aepted 11 August 215 First pulished online 23 Septemer 215) Astrt Oesity is one of the mjor helth prolems throughout the world. The present study investigted the preventive effet of epiltose rre non-digestile dishride on oesity nd metoli disorders in mie fed high-ft (HF) diets. Feeding with HF diets inresed ody weight gin, ft pd weight nd dipoyte size in mie (P < 1), nd these inreses were effetively prevented y the use of supplementl epiltose without influening food intke (P < 1). Cel pools of SCFA suh s eti nd propioni ids in mie fed epiltose were higher ompred with mie not reeiving epiltose. Supplementl epiltose inresed the expression of unoupling protein (UCP)-1, whih enhnes energy expenditure, to 2-fold in the gstronemius musle (P = 4) nd to 1 3-fold in the rown dipose tissue (P = 2) in mie fed HF diets. Feeding HF diets indued pro-inflmmtory mrophge infiltrtion into white dipose tissue, s indited y the inresed expression of monoyte hemotti protein-1, TNF-α nd F4/8, nd these inreses were ttenuted y supplementl epiltose. In differentited myogeni-like C2C12 ells, propioni id, ut not eti or n-utyri ids, diretly enhned UCP-1 expression y pproximtely 2-fold (P < 1). Tken together, these findings indite tht the epiltose-medited inrese in UCP-1 in the skeletl musle nd rown dipose tissue n enhne whole-ody energy expenditure, leding to effetive prevention of oesity nd metoli disorders in mie fed HF diets. It is suggested tht propioni id teril metolite ts s meditor to indue UCP-1 expression in skeletl musles. Key words: Epiltose: Propioni id: Oesity: Unoupling protein: Skeletl musle Non-digestile rohydrtes suh s dietry fires nd oligoshrides hve vriety of enefiil effets on our helth. Epiltose (4-O-β-gltopyrnosyl-D-mnnose) is rre nondigestile dishride, whih n e produed in onsiderle mounts y heting nd lkli tretment of ows milk (1). Reently, we hve developed method for the mss-prodution of epiltose using elloiose 2-epimerse (EC ) from the ruminl strin Ruminoous lus NE1 (2). Although our previous studies hve found tht epiltose enhnes the sorption of intestinl C nd Fe in rodents (3), informtion on its iologil funtions remins quite limited. As previous studies hve demonstrted tht some non-digestile rohydrtes meliorte metoli disorders suh s oesity, epiltose lso my hve potentil to prevent these disorders. Oesity nd relted diseses, suh s dietes nd CVD, hve emerged s mjor helth prolems in mny ountries, prtiulrly in the West (4). It is known tht oesity nd dietes re the results of omplex intertion etween geneti nd environmentl ftors. In prtiulr, the intke of ft-enrihed diet nd intke of exessive lories re primry ftors ssoited with the inresed ourrene nd further progression of metoli disorders. At the sme time, the onsumption of erels, mjor soure of non-digestile rohydrtes, is flling in Western ountries, nd numer of importnt reltionships etween the intestinl fermenttion of these rohydrtes nd the regultion of metoli disorders hve een suggested (5 7). It ppers tht the relese of teril metolites tht is, SCFA inluding eti, propioni nd utyri ids plys numer of importnt roles in metoli regultion. Previous studies hve shown tht luminl SCFA hnge the synthesis of gstrointestinl peptides suh s peptide YY nd glugon-like peptide-1, whih ontrol food intke nd gluose tolerne (8,9). In ddition, the SCFA trnsported to the irultory system reportedly t s regultors of energy metolism nd diposity (1,11). However, the preise roles of SCFA in the regultion of metoli disorders nd energy metolisms remin to e lrified. Energy metolism is orgnised y different systems or mehnisms in mmmls. The unoupling protein (UCP) is Arevitions: C, ontrol; Epi, epiltose; HF, high ft; MCP-1, monoyte hemotti protein; Std, stndrd diet; UCP-1, unoupling protein-1. * Corresponding uthor: T. Suzuki, fx , emil tkuy@hiroshim-u..jp Downloded from IP ddress: , on 17 Aug 218 t 23:31:1, sujet to the Cmridge Core terms of use, ville t

2 Epiltose prevents metoli disorder 1775 loted in the mitohondril inner memrne, where it unouples the oxidtion of fuels, minly ftty ids, from ATP prodution, so tht the energy ssoited with fuel oxidtion is simply relesed s het. UCP-1 is highly expressed in rown dipose tissue, whih speilises in the prodution of het through non-shivering thermogenesis (12). Therefore, UCP-1 tivtion ould serve s novel therpeuti pproh for the tretment of oesity. In ddition, reent studies hve demonstrted tht the etopi expression of UCP-1 in the skeletl musle of mie inreses whole-ody energy expenditure, resulting in the restortion of insulinemi, holestelolemi nd diposity (13,14). Although the sl density of UCP-1 in skeletl musle is lower thn tht in rown dipose tissue, evidene suggests tht inresed UCP-1 in skeletl musle ould mke signifint ontriution to whole-ody energy expenditure due to the lrge musle mss. The purpose of present study ws to investigte the preventive effet of epiltose on oesity nd metoli dysregultion in mie fed high-ft (HF) diets in reltion to intestinl fermenttion. Further, the diret effets of SCFA on UCP-1 expression in differentited myogeni-like ells were lso exmined. Methods Animls (Expt 1 nd 2) All niml studies were pre-pproved y the Hiroshim University Animl Committee, nd the mie were mintined in ordne with the Hiroshim University guidelines for the re nd use of lortory nimls (no. C11-24). Mle C57/BL6 mie (5 weeks old; Chrles River) were housed in ges in room with ontrolled temperture (22 (SEM 2) C), reltive humidity (4 6 %) nd lighting (light hours) throughout the study. The ody weight nd food intke of the mie were mesured every dy. The mie were llowed to limtise to the lortory environment with free ess to the ontrol diet (AIN-93G formul; Tle 1) (15) nd distilled wter for 1 week. In Expt 1, mie were rndomly divided into the following four groups; the stndrd diet (Std)-ontrol (Cont), Std-epiltose (Epi), HF-Cont nd HF-Epi groups (n 7 8 per group). The Std-Epi nd Std-Cont groups were fed diet ontining 7 % soyen oil with nd without 1 % epiltose y weight (Tle 1). The HF-Epi nd HF-Cont groups were fed diets ontining 7 % soyen oil nd 33 % lrd with nd without 1 % epiltose. Epiltose ws dded to the experimentl diets y sustitution for n equl mount of strh. The mie hd free ess to eh experimentl diet for 8 weeks. On dy 54 of the experimentl period, mie were fsted for 8 h, nd lood smples were olleted from the til vein to mesure plsm gluose, insulin nd TAG levels. At the end of the experiment, the mie were srified y exsnguintion under diethyl ether nesthesi. The livers nd ft pds (mesenteri, epididyml nd retroperitonel) were removed nd weighed, nd the epididyml ft pds were fixed with 4 % prformldehyde nd emedded in prffin for hemtoxylin eosin stining. Adipoyte size ws determined y verging the dimeters of t lest fifty dipoytes from the ft pd of eh mie. Tle 1. Composition of diets (Expt 1 nd 2) Ingredient Stndrd diet (g/kg diet)* High-ft diet (g/kg diet)* Mize strh Surose 1 1 Csein 2 2 Soyen oil 7 7 Lrd 33 Cellulose 5 5 Minerl mix Vitmin mix 1 1 L-Cystine 3 3 Choline itrtrte * Epiltose ws dded to diets t 1 % through sustitution of n equl mount of mize strh. Mize strh (α-mize strh; Chuou Shokuryo Co. Ltd). Csein (ALACID; New Zelnd Dily Bord). Crystllised ellulose (Ceolus PH12; Ashi Chemil Industry). Minerl nd vitmin mixtures were prepred ording to the AIN-93G formultion. The el ontents were lso olleted for orgni id nlyses s desried elow. In Expt 2, mie were rndomly divided into the following three groups: the Std-Cont, HF-Cont nd HF-Epi groups (n 7 8 per group). Experimentl diets nd periods were the sme s those in Expt 1. To evlute the effets of epiltose on lipid sorption nd exretion, fees smples were olleted ontinuously for 3 d from dy 51 fter the strt of feeding the test diets. At the end of the experiment, the liver, ft pds, gstronemius musle nd suprspulr rown dipose tissue were olleted nd weighed for quntittive RT-PCR nlysis s desried elow. Mesurements of plsm gluose, insulin nd TAG The plsm gluose nd TAG onentrtions were ssyed y enzymti methods using ommerilly ville kits (Gluose CII nd TAG E Test Wko; Wko Pure Chemil Industries). The plsm insulin onentrtion ws ssyed using n ELISA kit (Ultr Sensitive Mouse Insulin ELISA Kit; Moring Institute of Biologil Siene). Orgni id nlysis The el ontents were diluted with four volumes of distilled wter nd homogenised using polytron-type homogeniser. A 5 μl of 25 mm rotoni id s n internl stndrd ws dded to 7 μl of el ontents nd the mixture ws entrifuged t 15 7 g for 1 min t 4 C. The resultnt superntnt ws de-proteinised with 1 % sulphosliyli id nd diluted with two volumes of distilled wter. The smples were filtrted nd pplied to UPLC/MS system. Orgni ids (eti, propioni, n-utyri, iso-utyri, n-vleri, iso-vleri, lti, suini nd rotoni ids) were identified nd quntified using UPLC/MS system equipped with n eletri spry ionistion (ESI) interfe (Aquity UPLC; Wters Co. Ltd). The temperture of the pillry heter nd vporistion heter ws mintined t 12 nd 4 C, respetively. The flow rte of the sheth gs (N) ws 15 l/h. LC/ESI-MS ws rried out in sn mode from (m/z) +5 to 2 nd in seleted Downloded from IP ddress: , on 17 Aug 218 t 23:31:1, sujet to the Cmridge Core terms of use, ville t

3 1776 Y. Murkmi et l. ion reording (SIR) mode from (m/z) +59 for eti id, (m/z) +73 for propioni id, (m/z) +87 for n- nd iso-utyri id, (m/z) 11 1 for n- nd iso-vleri id, (m/z) 89 for lti id, (m/z) 117 for suini id nd (m/z) 85 1 for rotoni id. The UPLC system ws fitted with 1 8 μm C18 olumn (ACQUITY UPLC HSS T3, mm; Wters Co. Ltd) set t 4 C. Solvents A (wter formi id, 1: 1) nd B (methnol formi id, 1: 1) were run t flow rte of 2 ml/min using grdient from up to 5 % solvent B for 2 min, followed y % solvent B for 2 min, from 5 up to 6 % solvent B for 8 min, from 6 up to 8 % solvent B for 1 min nd from 8 up to 1 % solvent B for 2 min nd then redued linerly k to % solvent B over the next 1 min nd susequently mintined t the initil ondition for 5 min. The injetion volume ws 5 μl. Conentrtions of individul orgni ids were lulted from the pek re in the hromtogrm deteted with SIR reltive to the internl stndrd (rotoni id). Mesurement of fel TAG Fees smples olleted in Expt 2 were freeze-dried nd milled. Totl lipids were extrted from powdered fees (out 1 mg) using the Bligh & Dyer method (16). TAG levels in the extrts were estimted y n enzymti method s desried ove. Quntittive RT-PCR nlysis Totl RNA from the mouse tissues nd C2C12 ells were isolted using TRI regent (Sigm) nd reverse-trnsried using ReverTr Ae qpcr RT kit (TOYOBO) ording to the mnufturer s instrutions. Rel-time qpcr ws performed using Step One Rel-Time PCR system (Life Tehnologies) nd KAPA SYBR FAST qpcr kit (KAPA BIOSYSTEMS). The primer sequenes used for the PCR re shown in online Supplementry Tle S1. Dt were nlysed y the ΔΔC t method nd presented s fold hnges in gene expression fter normlistion to the internl ontrol (GAPDH gene expression level). Immunolot nlysis Mouse tissues (5 mg) were homogenised in 1 ml lysis uffer ontining 1 % (w/v) SDS, 1 % (v/v) TritonX-1 nd 1 % (w/v) sodium deoxyholte in 3 mmol/l-tris with protese nd phosphtse inhiitors (ph 7 4) using polytron-type homogeniser. Protein onentrtions were mesured using the BCA method (Piere Biotehnology). Tissue extrts were mixed with hlf volume of Lemmli smple uffer (3 onentrted) ontining 6 % (w/v) SDS, 3 % (v/v) glyerol, 15 % (v/v) 2-β-merptoethnol nd 2 % (w/v) romphenol lue in 188 mmol/l-tris, (ph 6 8) nd heted to 1 C for 5 min (17). Proteins (3 μg) were seprted y SDS-PAGE nd trnsferred to PVDF memrnes. Memrnes were lotted for UCP-1 nd β-tin using speifi ntiodies in omintion with HRP-onjugted nti-mouse or nti-rit IgG ntiodies. The lots were developed using the ECL hemiluminesene method (Perkin Elmer). Quntifition ws performed y densitometri nlysis of speifi nds on the immunolots using Imge J softwre. Cell ulture (Expt 3) To exmine the diret effets of orgni ids on UCP-1 expression in the skeletl musle, mouse myolsti-like C2C12 ell line ws used. C2C12 ells (CRL-1772; Amerin Type Culture Colletion) were propgted nd mintined using DMEM supplemented with 1 % FBS s desried previously (18). The ells were seeded into twenty-four-well pltes (1 9m 2 ; Thermo Sientifi In.) t density of ells/m 2. After the ells rehed onfluene, the medium ws repled with myogeni medium onsisting of DMEM supplemented with 2 % horse serum to indue myogeni differentition. All the experiments were onduted on dy 8 fter the strt of differentition. Cultures were used etween pssge 7 nd 11, nd the medium ws refreshed every 2 d. The experiment ws repeted three times, nd representtive nlysis is shown in the result setion. Three individul orgni ids eti, propioni nd n-utyri ids were dministered into the medium ( 3 3 mm-eti id, 1 1 mm-propioni id nd 1 1 mm-n-utyri id). These onentrtions were hosen sed on the physiologil levels present in the lood of rodents (19). The totl RNA nd protein ontents of ells inutedwithorgniidsfor24hweredeterminedtoexmine the UCP-1 gene nd protein expressions s desried ove. Sttistil nlysis All the vlues re expressed s mens with their stndrd errors. The signifine of differenes etween groups ws ssessed using the Tukey Krmer post ho test. A differene with P vlue < 5 ws onsidered signifint. Sttistil nlyses were performed using JMP softwre (version 12; SAS Institute In.). Results Feeding epiltose prevents high-ft-diet-indued metoli disorders (Expt 1) Feeding HF diets indued inreses in ody weight nd the weight of the three ft pds (epididyml, retroperitonel nd mesenteri dipose tissues, Tles 2 nd 3). The ody weight gin nd totl ft pd weights were inresed pproximtely 1 6-fold y HF diets (P < 1). Supplementl feeding with epiltose effetively prevented these inreses, nd the ody weight gin nd individul ft pd weights in the HF-Epi group were lower thn those in the HF-Cont group (P < 1) without ny differene in the food intke etween the two groups. There were no differenes in liver weight mong the four groups. Feeding epiltose did not hve ny effet on these prmeters in mie fed the stndrd diets. Plsm gluose, insulin nd TAG onentrtions in mie were mesured to exmine metoli dysregultion (Tle 4). Although no differenes were deteted in the plsm gluose onentrtion mong the groups, the HF diets inresed plsm Downloded from IP ddress: , on 17 Aug 218 t 23:31:1, sujet to the Cmridge Core terms of use, ville t

4 Epiltose prevents metoli disorder 1777 Tle 2. Body weight, food intke nd eum weight of mie (Expt 1) (Men vlues with their stndrd errors; n 7 8) Body weight gin (g/8 weeks) Food intke (g/8 weeks) Ceum tissue (mg/1 g ody weight) Ceum ontent (mg) Diet Men SEM Men SEM Men SEM Men SEM Stndrd Control Epiltose High ft Control Epiltose ,, Men vlues with unlike supersript letters were signifintly different (P < 5). Tle 3. Liver nd white dipose tissue weights (Expt 1) (Men vlues with their stndrd errors; n 7 8) White dipose tissue (mg/1 g ody weight) Liver (mg/1 g ody weight) Epididyml Retroperitonel Mesenteri Diet Men SEM Men SEM Men SEM Men SEM Stndrd Control Epiltose High ft Control Epiltose , Men vlues with unlike supersript letters were signifintly different (P < 5). Tle 4. Plsm gluose, insulin, TAG nd Homoeostsis model ssessment for insulin resistne (HOMA-IR) in fsted mie (Expt 1) (Men vlues with their stndrd errors; n 7 8) Gluose (mm) Insulin (ng/ml) TAG (mm) HOMA-IR Diet Men SEM Men SEM Men SEM Men SEM Stndrd Control , Epiltose , High ft Control , Epiltose , Men vlues with unlike supersript letters were signifintly different (P < 5). insulin onentrtions to 1 3-fold (P < 1), nd this inrese ws ttenuted y feeding epiltose (P < 1). Homoeostsis model ssessment for insulin resistne, n inditor of insulin resistne, in the HF-Cont group ws nerly doule thn tht in mie fed the stndrd diets (P < 1), ut the HF-Epi group showed mrked inrese in vlue similr to those of the Std-Cont nd Std-Epi groups. Plsm TAG onentrtions in the Std-Epi nd HF-Cont groups tended to e lower thn tht in the Std-Cont group, nd TAG onentrtions in the HF-Epi group ws 24 % lower thn tht in the Std-Cont group (P < 1). Hemtoxylin eosin stining of the epididyml ft pds showed tht feeding HF diets led to hypertrophy of the dipoytes, inditing exessive ft umultion in the ells (Fig. 1). The morphology of dipoytes in the HF-Epi group ws oserved to e similr to those in the two stndrd diet groups. The verge dimeter of dipoytes in the HF-Epi group ws pproximtely 3 % lower thn tht in the HF-Cont group (P = 3). Feeding epiltose leds to the umultion of orgni id pools in the eum (Expt 1) Intestinl teril metolites in the form of orgni ids were determined in the eum, s epiltose is known to exhiit preioti property. The levels of ll orgni ids mesured, exept for lti nd suini ids, were higher in the Std-Epi group ompred with the Std-Cont group (Fig. 2). Aeti, propioni, n-vleri nd iso-vleri id levels in the HF-Epi group were 3- to 6-fold higher ompred with the HF-Cont group (P < 1). Inrementl trends were oserved for suini, n-utyri nd iso-utyri ids y the supplementl feeding of epiltose in mie fed the HF diets. Downloded from IP ddress: , on 17 Aug 218 t 23:31:1, sujet to the Cmridge Core terms of use, ville t

5 1778 Y. Murkmi et l. (A) Stndrd diet High-ft diet (B) Dimeter of dipoytes (µm) Control Stndrd Epiltose Feeding epiltose enhned unoupling protein-1 expression in skeletl musle nd rown dipose tissue (Expt 2) To investigte the mehnisms underlying the epiltosemedited prevention of HF diet-indued metoli disorders, nother niml study using three experimentl groups (Std-Cont, HF-Cont nd HF-Epi groups) ws onduted, nd the expression levels of genes involved in rohydrte nd lipid metolisms together with energy expenditure in the liver, white dipose tissue, skeletl musles nd rown dipose tissue, were exmined. Feeding the HF diet inresed the ody weight nd ft pd weights in mie (P < 1), wheres supplementl feeding with epiltose prevented these inreses High ft Fig. 1. Hemtoxylin eosin stining imges nd dipoyte dimeters of the epididyml dipose tissue of mie in Expt 1. (A) Representtive hemtoxylin eosin stining imges of epididyml dipose tissue of mie re shown. A lk r indites 1 μm. (B) Quntifition of dipoyte dimeters is shown. Vlues re mens (n 7 8), with their stndrd errors represented y vertil rs., Men vlues with unlike letters were signifintly different (P < 5)., Control;, Epiltose. in similr mnner to the results oserved in Expt 1 (P < 1, Tle 5). No differenes were oserved in gstronemius musle or suprspulr rown dipose tissue weights mong the groups. The expression levels of two genes involved in ftty id synthesis ftty id synthse (FAS) nd etyl-coa roxylse-α were pproximtely 6 nd 4 % lower in the livers of the HF-Cont nd HF-Epi groups thn in those of the Std-Cont group (P < 1, Fig. 3). Gluoneogenesis-relted genes phosphoenolpyruvte roxykinse 1 nd gluose- 6-phosphtse were inresed 2- nd 3-fold, respetively, y feeding the HF diets (P < 1). Expressions of these genes in the liver were not influened y epiltose. In the pthogenesis of metoli syndrome nd oesity, mrophges re hemottrted y the monoyte hemotti protein (MCP)-1 to dipoytes, leding to inflmmtion. Although no differenes in lipoprotein lipse, hormone-sensitive lipse nd PPARγ gene expression levels were found mong the groups, the inflmmtion mrkers, MCP-1 (P < 1), TNF-α (P < 1) nd F4/8 expression levels (P = 4), were inresed 1 5- to 2 5-fold y feeding the HF diets (Fig. 4). Supplementl feeding with epiltose prevented the inreses in MCP-1 (P < 1) nd TNF-α expression (P < 1) nd tended to ttenute the inrese in F4/8. In ddition, UCP-1, Cide nd PPARγ otivtor 1α (PGC-1α) expressions in white dipose tissues were exmined, euse reent studies hve shown tht nother UCP-1-positive ell, referred to s eige dipoyte, resides in white dipose tissues nd ontriutes to the energy expenditure. However, no differene ws oserved in these two gene expressions in white dipose tissues (dt not shown). Among the genes exmined in the gstronemius musle, the expression of UCP-1, whih is involved in the dissiption of energy s het, therey enhning energy expenditure, ws influened y diet (Fig. 5), with UCP-1 expression in the HF-Epi group eing more thn doule ompred with the other groups t the mrna nd protein levels (P = 4). The rown dipose tissue speilises in generting het through non-shivering thermogenesis using UCP proteins. UCP-1 mrna nd protein expressions in the two HF diet groups were higher thn tht in the Std-Cont group (P < 1 nd P = 3), nd these UCP-1 expressions in the HF-Epi group were even higher thn tht in the HF-Cont group (Fig. 6). The UCP-1 expressions in the HF-Epi groups were pproximtely 25 % higher thn those in the HF-Cont groups t the mrna nd protein levels (P = 2 nd 3). The expression of PGC-1α, regultor of UCP-1 trnsription, in the HF-Epi group ws 5 % higher ompred with the expression levels in the other groups (P = 4). Fel exretions of TAG were higher in the two HF groups thn in the Std-Cont group (P < 1), ut feeding epiltose did not influene exretion (Std-Cont, 58 (SEM 6); HF-Cont, 2 16 (SEM 15); HF-Epi, 2 35(SEM 19) mg/3 d). No differene ws oserved in dry weights of fees olleted for 3 d mong groups. Propioni id indues unoupling protein-1 expression in differentited C2C12 ells C2C12 ells were used to exmine the diret effets of three mjor orgni ids eti, propioni nd n-utyri Downloded from IP ddress: , on 17 Aug 218 t 23:31:1, sujet to the Cmridge Core terms of use, ville t

6 Epiltose prevents metoli disorder 1779 (A) 1 8 Orgni id pools (µmol/whole eum) (B) ,,, Aette Propionte Ltte Suinte n-butyrte Orgni id pool (µmol/whole eum) Iso-utyrte n-vlerte Iso-vlerte Fig. 2. Orgni id pools in the eum of mie in Expt 1. Ceum smples were olleted t the end of experiment. (A) Pools of ette, propionte, ltte, suinte nd n-utyrte re shown. (B) Pools of iso-utyrte, n-vlerte nd iso-vlerte re shown. Std, stndrd diet; HF, high-ft diet; Cont, ontrol; Epi, epiltose. Vlues re mens (n 7 8), with their stndrd errors represented y vertil rs.,, Men vlues with unlike letters were signifintly different (P < 5)., Std-Cont;, Std-Epi;, HF-Cont;, HF-Epi. Tle 5. Body weight gin, food intke nd white dipose tissue, rown dipose tissue nd gstronemius musle weights (Expt 2) (Men vlues with their stndrd errors; n 7 8) Body weight gin (g/8 weeks) Food intke (g/8 weeks) White dipose tissue (mg/1 g ody weight) Brown dipose tissue (mg/1 g ody weight) Gstronemius musle (mg/1 g ody weight) Diet Men SEM Men SEM Men SEM Men SEM Men SEM Stndrd Control High ft Control Epiltose , Men vlues with unlike supersript letters were signifintly different (P < 5). ids on UCP-1 expression. The ells inuted with 1 nd 3mM-propioni id showed dose-dependent inreses in UCP-1 mrna expression (P = 3 nd P < 1, Fig. 7). Similrly, the UCP-1 protein expressions were inresed y 1 nd 3mM-propioni ids (P < 1). No signifint effet were oserved fter inution with eti nd n-utyri ids, lthough n inrementl trend ws oserved in the ells inuted with 1 mm-n-utyri id in omprison with the ontrol tretment. Downloded from IP ddress: , on 17 Aug 218 t 23:31:1, sujet to the Cmridge Core terms of use, ville t

7 178 Y. Murkmi et l. Disussion Although previous study demonstrted tht supplementl feeding with epiltose did not hve n impt on the plsm lipid onentrtion in rts fed high-surose diets (2), some preioti oligoshrides reportedly exhiit n meliortive effet on diet-indued oesity nd metoli dysregultion (9,21,22). The present study showed tht epiltose effetively prevents metoli disorders proly through the up-regultion of UCP-1 expression in skeletl musles nd rown dipose tissue in mie fed HF diets. Notly, our results revel tht propioni id, whih is produed in lrge mounts through the intestinl fermenttion of epiltose, indues UCP-1 expression in differentited myogeni ells. Oesity nd relted diseses re the result of omplex intertion of geneti nd environmentl ftors. Among environmentl ftors, ft-enrihed diet nd exessive energy intke re losely ssoited with the ourrene of these diseses, nd therefore the development of novel preventive pprohes hs een egerly ntiipted. Supplementl feeding with epiltose for 8 weeks resulted in signifint redution in ody weight gin nd viserl ft pd weight without ffeting Gene expression in livers (AU) FAS ACC-α PEPCK G6Pse Fig. 3. Expression levels of ftty id synthse (FAS) etyl-coa roxylse-α (ACC-α), phosphoenolpyruvte roxykinse 1 (PEPCK) nd gluose-6- phosphtse (G6Pse) in the liver of mie in Expt 2. Livers were olleted t the end of experiment. AU, ritrry units; Std, stndrd diet; HF, high-ft diet; Cont, ontrol; Epi, epiltose. Vlues re mens (n 7 8), with their stndrd errors represented y vertil rs., Men vlues with unlike letters were signifintly different (P < 5)., Std + Cont;, HF+ Cont;,HF +Epi. food intke or fel TAG exretion in mie fed the HF diets. The histologil nlysis of epididyml dipose tissues shows tht feeding epiltose suppresses the dipoyte hypertrophy indued y the HF diets. White dipose tissue stores redundnt energy in the form of TAG droplets during nutritionl exess, nd oesity is hrterised y n inrese in the size of dipoytes differentited from firolsti pre-dipoytes in dipose tissue. These findings suggest tht supplementl epiltose inreses whole-ody energy expenditure, resulting in lower ody nd ft pd weights. Severl studies hve demonstrted tht UCP proteins hve pthophysiologil roles in energy metolism in humn s well s in rodents (23,24). Our results suggest tht supplementl epiltose enhnes energy expenditure through inresed UCP-1 expression in skeletl musles nd rown dipose tissue. We foused on musulr UCP-1, in prtiulr, s skeletl musles mke up pproximtely 4 % of the totl dult ody weight in ontrst to the reltively smll ontriution mde y rown dipose tissue (25). It is likely tht whole-ody energy expenditure ould e signifintly inresed y the tivtion of musulr UCP-1. It hs een reported tht the overexpression of UCP-1 in the skeletl musle of mie inreses energy expenditure, resulting in deresed plsm insulin, gluose, nd holesterol levels s well s deresed diposity (13,14). In ddition, UCP-1 expression in the musles protets mie from HF diet-indued oesity (26). These findings re-infore our hypothesis tht the inreses in musulr UCP-1 mke signifint ontriution to the epiltose-medited prevention of oesity nd metoli dysregultion. The finding tht propioni id, t onentrtion orresponding to physiologil levels in the lood of humns nd rodents, stimultes UCP-1 expression in C2C12 ells suggests tht this SCFA medites the epiltose-indued inrese in UCP-1 in mouse musle. It hs een reported tht t lest two G protein-oupled reeptors (GPR) for SCFA, GPR41 nd GPR43 re expressed in musle ells nd C2C12 ells (27,28). Some studies hve demonstrted tht GPR41 nd GPR43 hve n importnt role in the regultion of energy expenditure nd rohydrte nd lipid metolisms (1,11). UCP-1 expression is inresed in C2C12 ells inuted with propioni id t 1 nd 3mM, nd smll, lthough NS, inrese is oserved in C2C12 ells inuted with.1 mm-n-utyri id. Funtionl Gene expression in dipose tissues (AU) , HSL LPL PPARγ MCP-1 TNF-α F4/8 Fig. 4. Expression levels of hormone-sensitive lipse (HSL), lipoprotein lipse (LPL), PPARγ, monoyte hemotti protein-1 (MCP-1), TNF-α nd F4/8 in the epididyml dipose tissue of mie in Expt 2. Epididyml dipose tissues were olleted t the end of experiment. AU, ritrry units; Std, stndrd diet; HF, high-ft diet; Cont, ontrol; Epi, epiltose; UCP, unoupling protein. Vlues re mens (n 7 8), with their stndrd errors represented y vertil rs., Men vlues with unlike letters were signifintly different (P < 5)., Std + Cont;, HF + Cont;, HF + Epi. Downloded from IP ddress: , on 17 Aug 218 t 23:31:1, sujet to the Cmridge Core terms of use, ville t

8 Epiltose prevents metoli disorder 1781 (A) Gene expression in gstronemius musle (AU) (B) UCP-1 protein in gstronemius musle (AU) UCP-1 UCP-2 UCP-3 PGC-1α LPL CPT-1α UCP-1 Fig. 5. Expression levels of unoupling protein (UCP)-1, UCP-2, UCP-3, PPARγ otivtor 1α (PGC-1α), lipoprotein lipse (LPL) nd rnitine plmitoyltrnsferse- 1α (CPT-1α) in the gstronemius musle of mie in Expt 2. Gstronemius musles were olleted t the end of experiment. (A) mrna expression levels of UCP-1, UCP-2, UCP-3, PGC-1α, LPL nd CPT-1α in the musle re shown. (B) The UCP-1 protein expression level is shown. AU, ritrry units; Std, stndrd diet; HF, highft diet; Cont, ontrol; Epi, epiltose. Vlues re mens (n 7 8), with their stndrd errors represented y vertil rs., Men vlues with unlike letters were signifintly different, P < 5., Std+ Cont;, HF+ Cont;, HF +Epi. (A) Gene expression in rown dipose tissues (AU) (B) UCP-1 protein expression in rown dipose tissues (AU) UCP-1 UCP-1 PGC-1α Fig. 6. Expression levels of unoupling protein (UCP)-1 nd PPARγ otivtor 1α (PGC-1α) in the rown dipose tissue of mie in Expt 2. Brown dipose tissues were olleted t the end of experiment. (A) mrna expression levels of UCP-1 nd PGC-1α in the rown dipose tissue re shown. (B) The UCP-1 protein expression level is shown. AU, ritrry units; Std, stndrd diet; HF, high-ft diet; Cont, ontrol; Epi, epiltose. Vlues re mens (n 7 8), with their stndrd errors represented y vertil rs.,, Men vlues with unlike letters were signifintly different (P < 5).,Std +Cont;,HF+ Cont;,HF+ Epi. ssys indite tht GPR43 is tivted y eti, propioni nd n-utyri ids to similr degree, wheres the rnk order of potenies for GPR41 is propioni > n-utyri eti ids (28). Aording to these studies, the propioni id-medited inrese in UCP-1 expression my our vi GPR41 tivtion. However, the underlying mehnisms for the epiltosemedited inreses in UCP-1 expression pper to differ etween skeletl musle nd rown dipose tissue, s feeding epiltose inreses PGC-1α, trnsription regultor of UCP-1, in rown dipose tissue only. Further studies re required to lrify the preise mehnisms for the epiltose-medited inrese in UCP-1 expression. A growing ody of evidene supports the notion tht oesity nd metoli disorders re hrterised y hroni stte of low-grde inflmmtion with pro-inflmmtory mrophge infiltrtion into white dipose tissue (29,3). This mrophge infiltrtion is triggered y the relese of pro-inflmmtory dipokines, inluding MCP-1, from lipid-lden dipoytes. One mrophges infiltrte into dipose tissue, they intert with dipoytes through TNF-α relese to inrese pro-inflmmtory dipokine seretion nd derese nti-inflmmtory dipokine seretion, leding to the progression of metoli dysregultion suh s insulin resistne nd hyperlipidemi. The inhiition of rosstlk etween dipoytes nd mrophges nd the derese in mrophge infiltrtion y feeding epiltose re onfirmed y the deresed expression of MCP-1, TNF-α nd F4/8 in dipose tissue. These results suggest tht supplementl feeding with epiltose prevents mrophge infiltrtion into dipose tissues due to the suppression of lipid umultion. Downloded from IP ddress: , on 17 Aug 218 t 23:31:1, sujet to the Cmridge Core terms of use, ville t

9 1782 Y. Murkmi et l. (A) UCP-1 gene expression (AU) Aeti id (mm) Propioni id (mm) n-butyri id (mm) (B) UCP-1 protien expression (AU) Aeti id (mm) Propioni id (mm) n-butyri id (mm) Fig. 7. Unoupling protein (UCP)-1 expression in C2C12 ells in Expt 3. Cells were olleted fter inution for 24 h with ette, propionte or n-utyrte. mrna (A) nd protein (B) expressions of UCP-1 in ells re shown. Dt re representtive of three independent experiments. Vlues re mens (n 6), with their stndrd errors represented y vertil rs., Men vlues with unlike letters were signifintly different (P < 5). AU, ritrry units. In onlusion, supplementl feeding with epiltose nondigestile dishride effetively prevents HF diet-indued oesity nd metoli dysregultion proly through inresed UCP-1 expression in the skeletl musles nd rown dipose tissue. Propioni id, n intestinl teril metolite, my e involved in the epiltose-medited inrese in UCP-1 expression in musles. Although further studies for the diret evlution of the inresed thermogenesis nd energy expenditure resulting from feeding epiltose re required, this non-digestile dishride ppers to hve the potentil s novel funtionl food for the prevention of metoli disorders. Aknowledgements The present study ws supported in prt y JSPS KAKENHI (T. S., grnt no ). JSPS hd no role in the design, nlysis or writing of this rtile. The uthors ontriutions re s follows: Y. M. nd T. S. designed nd onduted the reserh nd performed the sttistilnlysisofthedt;t.o.,w.s.,h.m.,h.m.nd S. T. helped in onduting the reserh; nd T. S. wrote the pper nd hd the primry responsiility for the finl ontent. All the uthors red nd pproved the finl version of the mnusript. The uthors delre no onflits of interest. Supplementry mteril For supplementry mteril/s referred to in this rtile, plese visit Referenes 1. Ctldi TR, Angelotti M & Bufo SA (1999) Method development for the quntittive determintion of ltulose in het-treted milks y HPAEC with pulsed mperometri detetion. Anl Chem 71, Ito S, Tguhi H, Hmd S, et l. (28) Enzymti properties of elloiose 2-epimerse from Ruminoous lus nd the synthesis of rre oligoshrides y the enzyme. Appl Miroiol Biotehnol 79, Suzuki T, Nishimuki M, Shinoki A, et l. (21) Ingestion of epiltose, non-digestile dishride, improves postgstretomy osteopeni nd nemi in rts through the promotion of intestinl lium nd iron sorption. J Agri Food Chem 58, Khn SE, Hull RL & Utzshneider KM (26) Mehnisms linking oesity to insulin resistne nd type 2 dietes. Nture 444, Johns DJ, Lindroos AK, Je SA, et l. (215) Dietry ptterns, rdiometoli risk ftors, nd the inidene of rdiovsulr disese in severe oesity. Oesity (Silver Spring) 23, Downloded from IP ddress: , on 17 Aug 218 t 23:31:1, sujet to the Cmridge Core terms of use, ville t

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