Effects of chronic cold exposure on succinate dehydrogenase activity in rat gastrocnemius muscle

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(1)Title. Effects of chronic cold exposure on succinate dehydrogenase activ ity in rat gastrocnemius muscle. Author(s). SZUKI, Junichi; GAO, Ming; OHINATA, Hiroshi; KUROSHIMA, Akihiro; KOYAMA,Tomiyasu. Citation. 冬季スポーツ研究, 2(1): 11-18. Issue Date. 2000-06. URL. http://s-ir.sap.hokkyodai.ac.jp/dspace/handle/123456789/6793. Rights. Hokkaido University of Education.

(2) 冬季スポーツ研究第2巻】号】1−柑．2000 （北海道教育大学冬季スポーツ教育研究センター紀要）. E付加tsofchromicco］de叩OSt］reOnSuCCjn且tedet．ydrogenaseactivity inratg且StrO川emiusmus亡Ie JunichiSUZUKlり，MingGAO2），HiroshiOHINATA3），AkihiroKUROSHIMA31andTomlyaSu KOYAMA之）〃エ虎口r血〃γす勘肝／∫P卸裏b由餅伽甜肝亡烏口門d血相〟ロ円仁ビ搾庸rカ「耶刑セr勘口巾．助丘血f血亡加ver雨中qr且血亡加叩山中卵〃却エ‘止βr‘地坪qrJ〕如ね山都加∫紺r仁み血Ff血Jピqr且J甜什馴鹿助f川呵肋た血f血亡加Ⅴ耶吋，訪甲α〃刃mピ苗rざJβ甲肝肋酎扉qrP卸扇bわ誹」∫加地7削コ几如拙丁αJCロJJ曙官，卸α〃. Abstract Theef托亡tSOf4−Week亡0）dexposureonsu亡dnatedehydrogenas巳（SDH）activityweresIud− ied jn gastro亡nemius mus亡1esin TTlale WistaT raTs．The Ta†s were randomlv dividedinto WarTn−aCC）imated印ntrOl〔WC，n＝］0）andcoId−aCClimated（CA，n＝TO）gTOuP5．Theratsor WCandCAgroupswerehousedat250cand50c，reSPe亡tiveEy，Bodyw巳ightatThetjTTleOf SaCrjfjcewassi邑nificantly）owerinCAthaninWC（P＜0・05）・Mus亡】ewe痩htsandmuscle We喧ht・TO−bodyweightraIioweres晦ni爪亡antty】owerinCAthaninWC（P＜0．05）．Indeep POrtionof．medialgastrocnemius（GASd），SDHactiv吋wass垣nif7亡antFyin亡reaSedafleTCO）d aecEimationintyp巳］，butnotintypeTTaandIIbfibers（P＜D．05）．TotalSDHa⊂tiv巾′（nor− malized fbr亡rOSS−SeCtiona］area）ofLGASd was sigrtificantFy greaTerin CAthanin WC （P＜0・05）・Mean円bercross−Se亡tionalareawassignjficant）ydecreasedaflercoldac仁【jmatioTl. by20％，18％，and17％，reSPeCTIvely，iTltyPeI，ⅠIa，and］lbfibers（P＜0．05）．Theseadaptive Changesprobablyimprovethe亡aPaCityofmus亡IetissuesIopromo†e†herTTlOgeneSisirtthe 亡01datmosphere．. Keywords：仁01dac亡Iimation；fibervpe；Skeleta】muscIe；SuCCinatedehydrogenase. Whenthehomeothermsar巳eXPOSedtoacold musclesthataremainlycomposedofoxidative atmosphere，thermogenesisintheskeletalmus− 茄bers，Howev巳r，flbertypespeeificchangesin Cles as wellasin other or各anSis enhanced. OXidativeenzymeactivityaftercoldac仁1imation. l，4．1D，lユ1. havenotyetbeendetermined．. Inhindlimbmusc）es，anincreaseintheoxi−. In this study，therefbre，We Observed the. dative enzyme a亡tlV］ty WaS Observed after. fibertyF）e亡OmPOSitionandsuccinatedehydro−. Chronic cold exposure7I21122）・In our previous. genase activity of亡aCh茄ber typein ske］etal. Study，SuCCinate dehydrogenase act）V］ty WaS musclesofratse叩OSedtocoldfbr4weeks，. increasedinsDleusanddeepportionofgastroc− nemiusmuscles，butnotinsupern亡ialportionof. M亡th（Ids. gastrocnemius rnus亡1e after cold acclimation. 〔50Cfbr4weeks）127．TheseresultssuggestthatA血仰山 anincreasein oxidative enzyme actlVlty WaS. TwenbTmaleWistarrats〔NipponSLCCo．Shi−. observed slfter coId acclimation in the skeEetar. ZuOka，Japan）wererandomlydividedintotwo. 11.

(3) Suzuk‖，GaoM，OhinataH，KリーoshjmaAandKtIyamaT. groups；Warm COntrOl〔WC；n＝10）and cold−. SDH actlVlty WaS meaSured usIng amOdifica−. acclimated group（CA；n＝10）．The rats ofCAtionofthe group w巳re eXPOSed to a coId temperature of. stalnlngPrOCedur巳descrjbedprevi− ously23）・Thismodificationin亡1udestheuseof. 5j＝lOC fbr4weeksfiomtheage of8weeks．8−dimethy］amino−2，3−benzophenoxazine TheWCratswerehousedat25土lOC．Therela− （meldolablue）insteadofl−methoxyphenazine. tivehumidityofboththecoldandwarmrDOmS. methylsulhte（mPMS）as the exogenous elec−. WaS about50％．Allrats were housed onepertroncaITier14），Sections10−Llm−thickwereincu−. Cageundera12hlight−darkcycle（1ight7：00− batedinamediumcontalnlnglOO mMphos− 19：00），and weregiven commerciaIlaboratoryPhatebufftr，PH7．6；柑％（W／V）poIyvinyIal−. Chow and tap water adIibi［um，The animals. COhol；0．75mMsodiumaヱid巳；0，1mMmeIdola. WereCaredfbrinaccordancewiththeMGuiding. blue；1，5mM nitrob］ue tetrazolium（NBT）；5. Princip］esfbrtheCareand Use ofAnimalsin. mM EDlÅ（disodiumsalt）；and48mM sueciT. the Field of Physiol亡唱ICalSciencesr’of the. nate（disodiumsalt）−Thenon−SPeC摘creduction. Physiologi亡alSo亡i叫OfJapan．. OfNBT was determined byin亡ubating serial sectionsintheabovem巳diumwithoutsuccinate．. 〃耶亡良川叩ね∫. Theimagesofincubateds巳Ctionsweredigitiヱed. Underpentobarbitalanesthesia（40mg几gi．p．），. Viaavid印SCannerattaehedtoalightmicro−. theleft gastrocnemius（GAS）muscle was ex− S亡OPe，andwerestoredoncomputerdiskwithin Cised，Weighed，andplacedinembeddingmeT 24h・The］ightsourceofthemi亡rOS亡OPeWaS dium，0・CLT・亡OmPOund（OPtimumco）dcutting. restrjcted to 570 nm（the peak absorbance. temperaturecompound；MilesInc．，Elkhart，IN， WaVelength ofdifbTmaZan；reaCtionindicator） USA）and rapid】y ftozenwithliquidnitrogen．by a narrow−PaSSinterftrence fi）ter（Asahi. Themusclesampleswerestoredat−20Bcuntil. SpectraCo・，Tokyo，Japan）・Thevideoim喝eOf. histochemicalanalyses．Inthisstudy，theImS−themusclesectionwascomposedofamatrixof. Cleswerenotfixedbefbreexcision．Becausethe. 640x4gO pixe］s（each pixe）had an area of. length ofmuscles af托亡tSthe measurementsof. O．503pmコ），The opticaldensity（OD）ofthe. fibercrossrsectionaIarea印，WeCannOtdirectIyimageswasana］yzedwiththeNIHimagepro− COmParetheresultsOfthepresentstudytothat. gram■ Using calibrated photographic density. Ofother studies usIng muSCle fixation．In the. SCa］e（GSR−2，RWInstitute ofCo］orandPho−. PreSentStudy｝allmusc］eSamP］eswerepreparedtography，Tokyo，Japan），the256possibIegray inthesamefhshion．We亡Ou）d，therefbre，亡Om−. levels were conv亡rted to OI〕units．From the. Parethevaluesofthenbercross−SeCtionaIarea. digitizedimage，the boundaries ofindividual. betweenWCandCAgroups．. musclefiberswereoutlinedandthentheavera− geOD fbrallpIXelswithintheoutlined nber. 〃f帥亡Jk脚加Jpr即ビ血rビ∫ノbr∫〟亡亡加α止血吋一. WaS detenTlinedL This averaglng PrOCedure re−. 血郡皿町印岬抽血坤. duced possib］e errors resulting ftom areas of. 1ヱ.

(4) St〕Hactivityaftcr亡0）dacc）iTT）ation. higher SDH−Stalnlng densltywithin a muscle. fh）mthemid−belly ofthe musclewithacryo−. 仙er糊．ThefinalODfbreachsectionwasde− tomeat−200C．Thesectionswerestainedfbrthe rived by subtract）ng the mean va）ue fbr the. myosinATPasemethod6）aflerpreincubationat. blank se亡tions（without substrate）n・Om the. pH4．6．Usingthismethod，muSC］巳flberswere. mean value fbr tissue sections with substrate. ClassifiedastypeI（dark），tyPeIIa（White），and. PreSent．IthasbeenreportedthattheODvalues. typeIIb（internlediate）in GASrnuSCles■The. fbr SDH activiqr determined by histochemical. imagesofincubatedsectionsweredigitizedvia. methodweredirectlyproportionaltothevalues. avideoscanner〔U−VPT−N，0）ympusCo・，To−. determinedbybiochemicalmethod（r＝0．99）16）. kyo，Japan）attachedtoalightmicroscope（BX−. AnestimateofthetotalSDHactivity（nonTlal− 50，01ympus），and. were stor巳d on亡Omputer. izedfbrcross−SeCtjonalarea）ofthemuscIewas disk，UsingafixedmicroscoplC magnincation，. deterrninedbasedonthemeanSDHa亡tivitiesofeachdigitizedimagehadanareaofO・15mm王▲ typel，lla，andIIb f拍ers，the mean cross− Fibercrossrsectionalareas were determined fbr SeCtionalareasofthesemusc）enbers，andtheir. 巳aChfibertyp巳uSingtheNIHimageprogram. r巳1ative proportions嘲，Th巳fbrmulaonwhi亡h. （WrittenbyWayneRasbandattheU．S，National. theestimatesoftotalSDHactivi吋WaSbasedis：. InslitutesofHeaIth），. （％亡Ontributionofeachfibertypetototalcross− SeCtionalarea）x（mean SDH activity ofeach. 伽血Jむ∬JJmI小一血. nbertype）．. Allre5Ultsar巳eXPreSSedasmeans土SE．Using theKolmogrov−Smirnofftest，Wefirsttestedthe. 仇細雨甜血軋叩皿血r甜ノbr町田加dm∫ビ. distribution of allparameters to deterTT．ine. α亡J血卸. Whetheritwascompatiblewithanormaldistri−. Transverse sections，10−Llm−thick，Were Cutbution．TheStudent’st−teStWaSuSedtoanalyze. m貼り・此ゆ〟門d〝闇亡JピW相加∫踊d椚比−CJだ明暗山一山一血中佃咄面相血川兢購. Initia］bodyweight Finalbodyweight GASweight GAS／bodyweightratio Group. （g）. （g）. （mg）. （m昌／g）. WC. 20乱9土ヨ．1. 284．8土4．7. 1ヰ5ヨ．6土41．6. 4．g7土0．11. CA. 20臥2±2．2. 252，9±2．医♯. l177．4士22．0＊. 4．59土0．04年. Va】uesaremeansj＝SE・WC，WaTl11COntrOl；C＾7亡0）d−aCClimatedLGAS，gaStTOCnemiusmusc［e・疇，SigniRcantlydi用汀ent斤omWCalP＜0．05．. 13.

(5) Suzuk‖，GaoM，OhjnataH，Kuro5hjmaAandKoyamaT. two−SamPle parametric comparisons．Non− 0 0. 0 4. Pararr］etric samples were ⊂OmPared bv the MaIln−WllitneyU−teStfbrtwo−Samplecompari−. sons，Di庁邑renceswerec亡InSideredtobestatisti＿. つJ. nV nU O. Ca11y51gnif紀a耐atP＜0．05．. Res山ts. 0 0 0 ∧ノー. 官主岳岳召虐UUS・SSOJU−山名已宏≡. □ wA ■ cA. TablelshowsthebodyandmuscIew巳ights Ofthetwogroups．Bodvweightatth巳timeof. SaCrificewassignifi亡antlv］owerinCAthanin WC（P＜0・05）・InGASmus亡1e，muSCIeweight and musc］e weight−tO−body weight ratio were. Type I TypeIIa TypeIIb. Fig．1M巳anflbercross−Se⊂tjonaEareaofeachf7beT typejTLdeepportionormedialgastrocnemiusTTluS亡1e． W亡，Warm⊂OntrOL；CÅ，COLd−a亡C）imated．A［ldataare representedasmean止SE・㌔Signif通nt）yd汗悔rent 斤omWC（P＜0．05）．. SjgnificantlyIowerinCÅthaninWC（P＜0：05）． Meanf沌ercrossrsectionalarea（FCSA）of． eachfiber年PeWaSShowninFig，l．lndeep. POrtionofmedialgastro亡nemius（GASd）mus− C］e，FCSAwasslgnificant）ydecreasedaftercoId √．ロ﹁X［。雇﹁凸0≡一て七白m〓︷蒜一望石ト. √blX［。篭＼□皇音宅昌凸S. ．ロ wCl■ cA. 0．0. TypeI TypeTIa TypeIIb Fi芭・ユMeansuccinatedehydrogenase（SDH）activitiy OreaChfibertypeindeepportionormedeal. Fig・3TロtalsuccjnatedehydTOgenaSe（SDH）activi坪. gastrocnemiusmuscle．GroupsarethesameasinFig． l．ALldataarerepresent巳dasmeans土SE．＋， Signjflcantlydjr托ren‖†omWC（Pく0．05）．. thesam巳aSinFig，連・A）LdataarerepreseTltedns TTlean吐SE・＊，S転njfi亡ant［ydif托rent什omWC. 14. 山deepportionormediaLgastTOCnemius・Groupsar巳（Pく0．05）.

(6) SDHa亡tiviTyaf【ercoldacclimation. nincantlyincreased after cold acclimation 〔P＜0．05），TotalSDH activity（norTna】ized fbr CrOSS−SeCtionalarea）ofGASdwassignin亡antly greaterinCAthaninWC（P＜0．05；Fig．3）．. Figure4shows the re］ationship between. relative SDHand FCSAinea亡hfibertypein 官主岳Jヱ岩層S・ⅥS呂JUq雲票≡. GASd．ThereIativeSDHactlVltyWaSObtained. bynormalizinganindividuaIflberSDHactiv和 O rI．2 0．4 口．古口．呂 1．0. 叫TypeIIa. basedonthefbllowIngCa）culation retativeSDH＝（Ⅹi−Ⅹminl／. 4UOO. （Ⅹmax−Ⅹmin）. Where Xiis SDH actlVlty Ofindividualfiber，. 3〔）口0. XminisminimumSDHactiviqrfbrallfibersin. 20亡I口. that musc）e，and Xmaxis maximum SDH acA. tivityfbrallf；b巳rSinthemuscleJnqrpeIfibers. 1（）00. WithlowerrElativeSDHactivity（rangeofO，20−. n. 0 （1．2 【I．4 （1．6 n．R l．O. qTypeIIb. 0．60），there was a marked decreasein FCSA anercoldacclimation．Incontrast，intypeITafi−. 400U. berswithhigherre）ativeSDHa亡tivity（rangeof O．50，0．90），there was a marked d巳CreaSein. ヨ00D. FCSA．＝0（10. Dis亡uSSi（ln. 】Of）0. 0. ローI．2 ロ．4 仁l．6 0．呂 1．O. RelativeSDHa亡tlV】ty. Thisstudyshowsthatadapt）VeChangesin the ske）etalmusc］e oxidative亡aPaCity，identi−. FiE．4 Histo呈TamSCOrrelat■ngre）a†ivesuccinate. fiedasanin亡reaSeinthe proportionoftypeI. dehydrenase（SDl・l）B．Ctivityandmeancross−S亡⊂tiona】 areaoFtyp巳Ⅰ（几），lla（B）and】1b〔C川bersin血ep. fibers andin SDH activityin that f弛er，Were. POHi即Orm亡dialgastrDCnemjusmuscle・Groupsare. Observedinratsexposedtocoldfbr4weeks，. thesameasinFig．1．AlldataaT亡rePreSentedas means±SE．. Inthisstudy，ratSWereeXpOSedtocoldfhr 4weeks．The same protocolfbrcoldacc］jma−. accljmation by20％，18％，and17％，reSPeC−. tionadoptedinthis study hasbe印uSed asa. tively，intyp巳l，Ha，andrIbf抽ers〔P＜0，05）．. Standard pro亡edure toinduee a physiologlCal，. Figure2showsthe mean SDH actlVlty Df. eSPeCialIymetabolica亡Climationto亡Oldimrats. each fibertypein GASd．IntypeI，butnotin ml”・151．H占rouxela［．（1956）observedastrong typelTaand TTb fibers，ST）rIactlVlty WaS Slg− Shivenng when warm−aC亡1imated rats were. 15.

(7) SuzukiJ，GaoM，OhinataH，KuroshimaAandKoyamaT. transftrredto60C，butno shiverlnga亡tlVltyln（Fig＿1）．Therefbreitis possible that th巳de−. thelegandbackmusclesofratsacclimatedto. CreaSein FCSAwithout anylnCreaSeinmito−. cold（6CC）fbr4−6weeks叩．This凸ndingsug− Chondrialvolume and／or number may a］so gests that，during 亡0Id exposure，Shivenng CauSetheincreaseinSDHactivity（i．e．，”con−. thermogenesis（ST）mayplayadominantroleinCentration”），Although the early phase ofcold acclimation，andthat. FCSA oftypeJfibers. WithlowerreIativeSDHactivitywasrnarkedly. non−Shivering thermogenesis〔NST）may be− decreased，FCSA ofthese nbers with higher COme mOre Predominantwithin 4−6 weeks． relativeSDHa亡tivitydidnot亡hangeafterco］d. Therefbre，Wethinkthatthe且daptlVe亡hanges. acclimation（Fig．4）．This result suggests that. observed in CA rats may be related with the. theincreasesin the size and／or the number of. increaseinoxidative metabolismsfbrSTinthe. mitochondriamay contribute mainly tothein−. earlystageandfbrNSTinthe］aterstageofcold. CreaSeinSDHactivityaftercoIdacclimation＿. acclimation．. Inthisstudy，FCSAofa11fibertypesin. In the present study，COld exposure slg− GAS muscles was slgnincantly smallerinthe. nincantlyincreased SDHactivityindeeppor−. COld−aCClimatedthaninthecontrolrats（Fig，l）．. tion of gastrocnemius（Fig．3）．nis董indingAreductioninFCSAmaybecausedbymusc）e SuPPOrttheresultsofpreviousinvestlgationsin. atrophybecause，inbothSOLandGASmus亡1es，. back muscles71andin hind】imb muscleslu2） the muscl巳Weight−tO−body weigllt ratio was Harrietal▲〔19糾）havereportedthattheactiv−. Signifi亡ant］y］essinCAthaninWC（Tablel）．. itiesofSDH，Citrat亡Synthase，andcytocromec The reductionin FCSA aft巳r COld acclimation OXidasedidnotchangeanercoldexposure（5QC. hasbeenobservedbyotherinvestlgatOrS川・201. fbr6weeks）in rat gastro亡n巳mius muscle91．. A］though the cause fbr the reductionis un−. However，they measured these enzyme activ− kJtOWn，it seems possjble that anin亡reaSein. itiesbybio亡hemicalmethodusinghomogenate CircuIatlngStreSShormonescausedbycoldex− musc）esamples．lnthepresentstudy，increasedposure contributes partly to the reduction in. SDHactivltyWaSObs亡rVedonlyintypelfibers FCSA．Chrc．nica）Iy. elevated plasmalevels of. （Fig・2），Whichcomposelesspartofgastrocne− glucocorticoidmay causesk巳】eta］musc］eatroq. miusmusclethanotherfiberbrpesコl．Ttisthere− phy嘲・In rats，P］asma corticosteronelevels fbrepossiblethatanincreaseinoxidativeen−. Wereelevatedafler7daysofchroniccoIdexpo−. ZymeaCtjvitiesa鮎r仁01dacclimationisunder− Sure（4中C），andreturnedtobaselinewithin14. estimatedwhentheseactivitiesaremeasuredby. daysofexposure5）．Fukuharae［a］．（1996）re−. biochemica】method．Theincreasesin the size PDrtedthattheincreasedplasmacorticost巳rOne. and／Drthenumb巳rOfmitochondriamay cause levelscausedbycoldexposure（T30c）陀tumed theincreasein SDH actlVIty，ln the present. tonormaIleveIswjthin5daysofexposurein. Study，rnean fiber cross−SeCtionalarea（FCSÅ）. rats問■ltistherefbrepossiblethat，inthepre− Sent Study，Plasma corticosteronelevels were. WaS Slgnincantly decreasedin a）Ifiber types 16.

(8) SDHELCtiYityaf［ercoEdacctimation. elevated during atleastearly stage of4week. to coldin F344rats．AmJPiuJSfo12甜. COlde叩OSure，andcausedthereductioninmus−. 岬ビ印加け町血官許血ve Cロ〃甲P加ゎJ3丁）＝. CleFCSA．. R弓lヨ1−Rウ41．. In亡OnClusion，Wehaveshownthatanin− 5．BIigh−Tynan ME，Bhagwat SA，and Cas−. CreaSeinSDHactivityoftypeI茄beroccurred. tonguayTW（1993）The efftctsofchronic. in the skeleta］muscles ofrats cold−aCClimated. COld亡XPOSureOndiurnalcorti亡OSterOneand. fbr4weeks・Theseadapt】VeChangesprobably. aldosteronerhythmsinSprague−Dawleyrats．. improvethecapaciqTOfmuscletissuestopro−. P匂′由‖お厄v54：3后ヨーヨ67．. motethermogenesisinthecoldatmosphere．. 6．BrookeMHandKaiserKK（1970）Muscle fiber types：How many and what kind？. A仁山owledg亡m亡nt. 」r亡南〃占王什OJ王3：36ウー37ウ．. 7．Dauncy MJandlngram DL（19悶）Influ−. This study was paIlially supported by a. enceofenvironm∈ntaltemperatureanden−. Grant−in−Aid fbr Scientific Research fhmthe. ergyintake on skeletalmus亡Ie resplratOry. Ministry ofEducation，Scienceand Culture of. enzymes and morphoIogy，EurJ如J. JapanO呂7呂003呂．. P加ゎJ粥：23ウー244，. 8，FukuharaK，KvetnanskyR，CizzaG，Pacak K，Ohara H，Goldstein DS，and KopimIJ. Refヒren亡e旦. （1996）Interrelations between sym− 1，Ad岳n C，ArdevoIA，RemesarX，A）emany M，andFem畠ndez−L如ezJA（1995）Hind］eg muscle energy and substrat亡balan亡eSin. Pathoadrenalsystemsinratsexposedtocold stress．J〃b〟r亡把〃由仁r山口J8：5ココー54l．. 9，Harri M，Dannenberg T，Oksanen−Rossl，. COId−eXPOSed rats．］軸Bio11！帽：1243−. HohtolaEandSundinU（19＄4）Re］atedand. 125l．. unrelated char唱亡Sin response toeXerCise. 2・ArmstrongRB，andPhelF）SRO〔19B4）MusT. Cle fiber type composition of the rat hindlimb．Am．］Ana（17l：259−272， 3．Aquin L，Lechner AJ，Sillau ＾H，and. and coIdin rats：a reeVa）uation．JApp］邦岬血相7：14g9−1497． 10LHiroux O，HartJS，and Depocas F（1956）. Metabolisrnandmuscleactivityofanesthe−. Ban亡hero N（19帥）Analysis ofthe shape. tized warm and cold ac亡Iimated rats on exr. Changes ofmusc）e抗ber cross sectionsin. POSuretOCO】d．ノノJ即ノア桓・油川：ヨ99−403．. guineapigsraisedat220Cand5ロC．即i］gers Jrcム385：223−22B． 4．Baba］donAM，Florez−Duquet ML，Hami1−. tonJS，McDonald RB，and Horwitz BA. 1l．H卓roux O，andPierreJSt（1957）Efftctof. COIda仁亡1imationonvascu］arizationofears，. heart，】iver，andmusc］esofwhit亡ratS．AnJ Physio＝朋：1日−1占乱. （1995）Efftctsofageandgenderonbrown 12．Hi止sonRC，WegrzynLE，OsborneDF，and fhtandskeletalmusclemetabo］icresponses KarllE（1996）Alanyl−glutamine. prevents. lT.

(9) SuzukiJ，GaoM，OTlinataH，Kuro与himaAandKoyamaT. muscle atrophy and glutamine synthetase. 21．SuzukiJ，GaoM，YahataT，KuroshimaA，. inductionbyglucocorticoids．AmJ. andKoyamaT（1997）Capillarygeometryin. 271（加即血叫卜血一昭血涙亡加甲剖如混. thesoleusmus亡1eofFatS亡01d−aCClimatedfbr. 40）：Rl165−Rl172．. 68generations．Acfa PJvsio［Scand160＝. 13．JanskyL（1973）Non−Shiveringthermogene−. Sis andits themoregulatory slgnificance．. 24ユー250．. 22，Su孔IkiJ，GaoM，OhinataH，KuroshimaA and KoyamaT（1997）Chronic亡0］d expo−. β由J尺ピV4＄：85−1〕2．. Sure Stimu］ates microvscular remodelling. 14．KuglerP and WrobelKH（1987）M巳1do］a Blue：A new electron carTier fbr the hist〔ト. Preftrential】y］n OXidative musclesin rats・. 亡hemicaldemonstration ofdehydrogenases. 舟〃Jf＞わ岱如才（inpress）・. （SDH，LDH，G−6−PDH），His［ochemis！Ty59：. 23．Van Noorden CJF and Fr・ed巳riks WM. 97−10ウ．. 15．Kt］rOShimaAandYahataT（1985）Efftctof. tory manualofcurrent methods−Micro−. fbodrestrictiononcoldadaptabiIityofrats・. SCOPyhandbooksVol．26，Oxfbrduniverslty. CanJf坤γ∫由J朗αr椚即OJ椙：占告−71．. PreSS，NewYork▲. 16．Martin TP，Vai）asAC，DurivageJB，Edg− ertonVR，andCastlemanKR（1985）Quan−. titativehistochemicaldeterminationofmus− Cle enzymes：Biochemicalverification．J 〃加f班ビmq血亡鮎m33：105ヨー1059． 17．Mathieu−CosteIIo O（1987）Capillarytortu−. OSityanddegreeofcontractionorextension ofskeletalmuscles．A4icrovasc Res33：9B− 117． 18．SiekGC，SacksRD，Blanco CE，and Edg−. erton VR（1986）SDH activityand亡rOSS− sectionalarea of muscle朽bersin cat dia− phragm．JA即JP如io［60：12糾−1292．. 川．Siek GCLewis MIand Bはnco CE（1989） E能ctsofundernutritjonondiaphragm甫ber Siヱe，SDHactivlty，andfhtigueresistance．J 』即けわ′血J砧：2196−2205． 20，Si】】auAH，AquinL，LechnerAJ，BuiMV， and BancheroN（19帥）Increasedcapillary. SupPlyinskeletalmuseIeofguineaplgSaC− CIimatedtocold．RejPP桓′Sio14王：233−245．. 18. （1992）Enzyme Histochemistry−Alabora−.

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