Protein metabolism in the pectoralis muscle and liver of hibernating bats, Eptesicus fuscus

Transcription

1 J Cmp Physil (1983) 152:13~144 Jurnal f Cmparative Physilgy. B 9 Springer-Verlag 1983 Prtein metablism in the pectralis muscle and liver f hibernating bats, Eptesicus fuscus Marshall E. Yace* Divisin f Bilgical Sciences, University f Michigan, Ann Arbr, Michigan 48109, USA Accepted April 26, 1982 Summary. Seasnal variatins in prtein metablism f the pectralis muscle and liver f the big brwn bat, Eptesicus fuscus, are examined in relatin t seasnal changes in physilgical status. A technique is described fr the determinatin f prtein synthetic rates in viv in animals t small fr cnventinal methds. The results indicate n detectable rates f prtein synthesis in hibernating bats during trpr buts (Table 2). Rates f synthesis in hibernating bats during perids f arusal are cmparable t thse f active summer bats (Table 2), despite the fact that the hibernating bats had nt eaten in ver 2 mnths. Rates f prtein degradatin were calculated frm the rate f urea frmatin in trpid bats (Figs. 4, 5), the verall lss f pectralis muscle and liver prtein mass during hibernatin (Table 3), the prprtin f the ttal time f hibernatin spent in trpr and arusal (Table 1), and the bserved rates f prtein synthesis (Table 2). These estimates (Table 4) indicate negligible rates f prtein degradatin in trpid bats. Hwever, prtein degradatin during peridic arusals is cmparable t that f summer bats after an vernight fast. These findings are cnsistent with earlier bservatins suggesting that significant glucnegenesis frm tissue prtein ccurs during spntaneus arusals frm hibernatin. Intrductin The big brwn bat, Eptesicusfuscus, is a cmmn insectivrus species ranging thrughut much f * Present address: Marine Bilgy Divisin, A-002, Scripps Institutin f Oceangraphy, University f Califrnia San Dieg, La Jlla, Califrnia 92093, USA the cntinental United States and suthern Canada. These bats d nt underg seasnal migratins (Beer 1955). Rather, they hibernate lcally during winter, even at the nrthern end f their range, where the first and last killing frsts may be separated by as much as six mnths (see Beer and Richards 1956). Hibernatin is thught t be a perid f ttal fasting fr these and ther insectivrus bats since flying insects are scarce r absent during winter mnths and bats d nt cache fd. The energetic requirements f E. fuscus during hibernatin are met primarily by the xidatin f fat reserves which were accumulated the previus fall (Beer and Richards 1956; Yace 1983). Hwever, E. fuscus als experiences significant decreases in lean mass, pectralis muscle mass, and ttal pectralis muscle prtein, indicating significant xidatin f tissue prtein during hibernatin (Yace 1983). Similar relative decreases in lean mass and ttal bdy prtein have als been bserved in hibernating arctic grund squirrels (Galster and Mrrisn 1976). The bservatin that hibernating mammals appear t xidize tissue prtein is nt surprising in light f the fact that net prtein catablism appears t be a universal feature f fasting in nnhibernating mammals (see Felig 1979) and birds (see Le Mar et al. 1981). Hwever, these bservatins pse the questin f hw prtein metablism is regulated during hibernatin since excessive degradatin f tissue prtein culd cmprmise the ability t fly, a lethal cnditin fr an animal which must fly t feed. The present study examines prtein metablism in hibernating bats in relatin t the patterns bserved in fasting nnhibernating mammals. Prtein metablism underges time-dependent changes during fasting in nnhibernating mammals. During shrt-term fasting (1-2 days) the rate f prtein degradatin remains the same r is increased rela-

2 138 M.E. Yace: Prtein metablism in hibernating bats tive t that f the fed state, whereas synthetic rates drp significantly (see Millward and Waterlw 1978; Li et al. 1979), leading t a rapid lss f ttal bdy prtein (see Gdman and Ruderman 1980). The amin acids released frm degraded prteins are used in synthesis f ther prteins r they are xidized, either directly, via the citric acid cycle, r indirectly, via glucnegenesis. As fasting prgresses (in animals capable f prlnged fasting), glucnegenic demand decreases gradually and there is a prgressive decrease in the rate f net prtein lss until fat reserves are depleted (see Cahill 1976; Le Mar et al. 1981). Prtein synthesis als slws dwn as fasting prgresses (Millward and Waterlw 1978). The calric equivalent f the amunt f ttal bdy prtein lst by E. fuscus during 4 mnths f hibernatin crrespnds t apprximately 10% f the energetic expenditure during that perid (calculated either as a fractin f the ttal calric cntent f the cmbined decrease in prtein and fat mass r as a fractin f the estimated ttal metablic expenditure [see Yace 1982]). This prprtin is much lwer than that bserved in nnhibernatrs in shrt-term fasting, but is similar t that f bese humans (see Felig 1979) and bese geese (see Le Mar et al. 1981) after prlnged fasting. Therefre, the verall lss f prtein relative t verall metablic expenditure during hibernatin in E. fuscus is similar t that in prlnged fasting f nnhibernating mammals, suggesting that prtein catablism may decrease during hibernatin as it des during prlnged fasting. Hwever, hibernatin differs frm prlnged fasting in that it is nt a unifrm physilgical state. Hibernatin cnsists f a series f trpr buts, during which bdy temperature is allwed t fall t ambient levels and metablism is very lw. Trpr buts accunt fr virtually all f the time in hibernatin, but less than 20% f the metablic expenditure (Wang 1978; Yace 1982; Table 1). Trpr buts are separated by peridic arusals, during which bdy temperature is briefly restred t nrmthermic levels. Arusals accunt fr very little f the time, but ver 80% f the metablic expenditure f hibernatin (see Wang 1978 ; Yace 1982; Table 1). In view f the metablic hetergeneity f hibernatin, tw distinct patterns f prtein metablism culd accunt fr the lw verall rate f prtein lss bserved in E. fuscus: (1) hibernatin culd resemble lng-term fasting, with a prgressive decrease in the rates f prtein synthesis and degradatin r (2) the lw rate f prtein lss culd result primarily frm a very lw rate f prtein degradatin during trpr buts, with little r n time-dependent change in the rate f degradatin in either the trpid r arused states. The present study attempts t evaluate these tw pssibilities. Materials and methds Animals. Bats were captured in barns and attics in the vicinity f Ann Arbr, Michigan. ' Summer' animals were captured in their day rsts, held vernight at rm temperature, and used the fllwing day. 'Hibernating' bats were captured in late September and early Octber, when they were quite fat, but still ccasinally active. They were induced t hibernate in the labratry in a darkened chamber at 5-7 ~ and apprximately 100% relative humidity, cnditins apprximating thse f natural hibernacula (Beer and Richards 1956). The bats used fr prtein synthesis measurements were hused tgether in a large cage. The remaining bats, which were used t determine the frequency and duratin f trpr buts and arusals and plasma and urinary urea levels, were hused individually in negalln paint cans fitted with a cardbard structure int which they culd climb and hang. A cpper-cnstantan thermcuple was attached t this structure t prvide a cntinuus recrd f air temperature inside the can. Dampened wd shavings prvided a water-saturated atmsphere. Trpr buts and arusals. Can and cld rm temperatures were recrded cntinuusly during Nvember and December, 1981, fr a ttal f 13,632 h f bservatin. When bats were trpid, the can temperature was indistinguishable frm that f the cld rm. When they underwent spntaneus arusal, the can temperature became elevated apprximately 5 ~ relative t the cld rm, allwing easy determinatin f trpr but duratin, arusal frequency, and arusal duratin. Plasma and urinary urea. Bats which had been trpid fr varius intervals were remved frm their chambers and sacrificed by decapitatin. Bld was cllected in heparinized tubes and centrifuged at tp speed in a clinical centrifuge. The plasma was transferred t plastic vials and frzen n dry ice. Urine samples were drawn directly frm the bladder with a Hamiltn syringe and urine vlume was nted t the nearest micrliter_ The samples were then transferred t plastic vials and frzen n dry ice. All samples were stred at -70 ~ until analysis. Five animals were induced t aruse frm trpr by remving their cans frm the cld rm and allwing them t remain undisturbed fr ne hur at rm temperature. During this time all five bats arused and remained nrmthermic (bdy temperature = 33.4_+ 1.4 ~ range = ~ At the end f the hur the bats were sacrificed and samples were taken as abve. All five bats had urinated during this perid, but the urine was nt cllected. The urea cncentratin in plasma and urine samples was assayed by the cupled ureaseglutamate dehydrgenase methd (Sigma tech. bull. n. 65-UV). Prtein synthesis. Freshly captured summer bats were fasted vernight and used the fllwing day. Winter bats were held in hibernatin fr tw mnths prir t use. After this perid they were remved frm the envirnmental chamber and allwed t aruse undistm'bed fr apprximately ne hur. During this t~me the bats achieved bdy temperatures f ~ Fifteen f these bats were used immediately. The remaining eight were returned t the chamber vernight. All f these animals had re-entered the trpid state by the fllwing day when they were used in prtein synthesis experiments. Since hiberna-

3 M.E. Yace: Prtein metablism in hibernating bats 139 trs are refractry t external stimuli during the early part f a trpr but (Galster and Mrrisn 1975), this prtcl ensured that all f the trpid bats were in a refractry state, making it pssible t handle and inject them withut inducing arusal. Prtein synthesis was measured using a methd based n that f Garlick et al. (1980). In all cases the bats were given an intraperitneal injectin (0.01 ml/g bdy mass) f 150 mm phenylalanine in 0.9% NaC1, labelled with 50 gci/ml L-[4-3H]- phenylalanine (New England Nuclear). Iutraperitneal injectin was necessary because these bats are t small t be reliably injected intravenusly. After injectin, summer bats and bats arused frm hibernatin were allwed t rest undisturbed at rm temperature and trpid bats were allwed t rest undisturbed at 3 ~ prir t sacrifice. Initial samples were taken 10 min after injectin t allw the label t equilibrate with plasma and intracellular amin acid pls (see Figs. 2, 3). Bats were sacrificed at the designated times by decapitatin and bld was cllected in heparinized tubes n ice. Then the pectralis muscles (bth sides) and liver were rapidly excised and frzen n dry ice. The bld samples were centrifuged fr 10 min at tp speed in a clinical centrifuge. Plasma samples (50 Ixl) were cllected and frzen n dry ice. All samples were held at -70 ~ prir t analysis. In summer bats and arused bats bld samples were cllected at 10, 20, and 30 rain after injectin. Samples f pectralis muscle and liver were frzen apprximately 2 and 3 min, respectively, fllwing sacrifice. Trpid bats were sacrificed and bld samples were taken 10 and 120 rain after injectin. Muscle and liver samples were frzen apprximately 2 and 3 min, respectively, after sacrifice. Since the rate f prtein synthesis is calculated frm the change in specific activity f prtein-bund phenylalanine between 10 and 30 min (fr nrmthermic bats) r between 10 and 120 min (fr trpid bats) all data are reprted at the nminal times f 10, 20, 30, and 120 min. Sample preparatin. The muscle and liver samples were sliced with a razr blade while still frzen and rapidly hmgenized in 3 ml f ice-cld 2% HC104 using a Tekmar Tissumizer set at tp speed. Prtein was remved frm the plasma samples by similarly hmgenizing 50 gl f plasma in 3 ml f ice-cld 2% HCIO 4. These hmgenates were centrifuged fr 10 rain at tp speed in a clinical centrifuge. The supernatants were decanted int fresh tubes t which 1.5 ml f a saturated triptassinm citrate slutin was added (t precipitate the HC104). This preparatin was centrifuged again at tp speed in a clinical centrifuge fr 10 rain and 1 ml f the resulting supernatant was used fr the determinatin f the specific activity f free phenylalanine in plasma, muscle, and liver. The HC104 precipitates f muscle and liver prtein were washed three times by resuspensin in 5 ml f 2% HCIO4, fllwed by centrifugatin at tp speed in a clinical centrifuge fr 10 rain. The prtein in the final precipitate was hydrlyzed in 5 ml f 6 N HCt fr 24 h at 110 ~ These samples were then evaprated t dryness and resuspended in 3 ml f 0.5 M sdium citrate, ph 6.3. A 1 ml sample f this prtein hydrlysate was used fr determinatin f the specific activity f prtein-bund phenylalanine in muscle and liver. Specific activity f phenylalanine. Accurate determinatin f specific activity depends n the islatin f phenylalanine. The prcedure used here (based n Garlick et al. 1980) invlves the enzymatic decarbxylatin f phenylalanine t fl-phenethylamine (PEA) and the subsequent extractin and assay f PEA specific activity. Aliquts (1 ml each) f the supernatants frm plasma, muscle, and liver samples, f the hydrlysates f muscle and liver t < UJ 13_ > 0 EE c I NaOH Cncentratin (M) c ~3 " < LI_._c Fig. 1. The effect f NaOH cncentratin n the recvery f phenethylamine (PEA). The islatin f PEA is based n extractin int the rganic phase at basic ph and subsequent extractin int the aqueus phase at acidic ph. Preliminary experiments shwed that the recvery f PEA was maximized by snicatin (fr thrugh mixing) and by adjustment f the NaOH cncentratin used in the first extractin t 0.5 M. The prcedure described in the Materials and methds sectin resulted in ver 90% recvery f bth PEA and phenylalanine standards prtein, r f a standard slutin f 20 I~M phenylalanine in 0.5 M sdium citrate were incubated vernight in capped tubes at 37 ~ with 0.5 ml f a suspensin f 0.1 U f phenylalanine decarbxylase (Sigma, P 2626) in 0.5 ml sdium citrate, ph 6.3, cntaining 0.5 mg/ml pyridxat phsphate as a cfactr. Under these cnditins virtually 100% f the phenylalanine is cnverted t PEA. PEA was then extracted and assayed using a mdificatin f the prcedure f Suzuki and Yagi (1976), as fllws. After enzymatic cnversin t PEA, the samples were centrifuged fr 10 rain at tp speed in a clinical centrifuge t pellet the enzyme. The supernatants were decanted int 30 ml Crex centrifuge tubes and I ml f 0.5 M NaOH (~.5 ml fr hydrlysates) and 10 ml chlrfrm/n-heptane (1:3, v/v) were added (see Fig. 1). This mixture was snicated fr 15 s at 40 W using a Bransn Snifier and then centrifuged fr 10 min at 3,000 g t reslve the phases. The cntents f each tube where then transferred t a separatry funnel, the aqueus (bttm) phase was discarded, and the rganic (upper) phase was returned t the centrifuge tube. Then 5 ml f chlrfrm and 4 ml f 0.01 M H2SO ~ were added and the mixture was snicared and centrifuged as abve. A 1 ml sample (2 ml fr hydrlysates) f the aqueus (upper) phase was remved fr liquid scintillatin cunting in 10 ml f a xylene based scintillant (Fricke 1975). A further 1 ml aliqut (20 gl fr hydrlysates) was analyzed fr PEA cncentratin accrding t Suzuki and Yagi (1976). Calculatins and statistics. The rate f prtein synthesis was calculated frm the specific activities f free (S,) and prteinbund (SD) phenylalanine. The rati f SJS~ was calculated fr each muscle and liver sample. Then the simple linear regressin f [(Sb/S~) x 100] n time since injectin (in days) was calculated. The slpe the standard errr f estimate f the regressin line was used here as an estimate f the fractinal rate f prtein synthesis (%/day). The rates f prtein synthesis in summer and winter bats were cmpared using analysis f cvariance. Null hyptheses were rejected at the 0.01 level...3 < ta3 13-

4 140 M.E. Yace: Prtein metablism in hibernating bats Since there is n straightfrward way t measure prtein breakdwn in viv, rates f prtein breakdwn were calculated frm the rates f synthesis and the net change in liver and muscle prtein mass during hibernatin accrding t the frmula: FBR = FSR + FNL where FBR is the fractinal rate f prtein breakdwn (%/day), FSR is the fractinal rate f prtein synthesis (%/day), and FNL is the fractinal rate f net prtein lss (%/day). Values fr FNL in trpid bats were calculated frm rates f urea accumulatin (Figs. 5, 6). Values fr FNL in hibernating bats during spntaneus arusal were calculated as the difference between the verall decrease in prtein mass f pectralis muscle and liver during hibernatin (Table 2) and the amunt which culd be attributed t prtein lss in trpr [calculated frm FNL in trpr and the fractin f the ttal time f hibernatin spent in trpr (Table 1)]. It was assumed that the fasted summer bats were in negative nitrgen balance, therefre FBR > FSR. The ptential fr errr is cnsiderable and nt quantifiable in the estimates f FNL in hibernating bats during spntaneus arusal and in fasted summer bats. Therefre, althugh numerical values are assigned t FBR, they are used nly t make qualitative cmparisns. Results Trpr buts and arusals Hibernatin in E. fuscus cnsists f a series f trpr buts averaging h in duratin, separated by perids f arusal averaging h (Table 1). The duratin f trpr buts (range = h) and arusals (range= h) bth vary cnsiderably. Of the 13,632 h f bservatin reprted here, 98% f the time (13,355 h) was spent in trpr. The remaining 2% (277 h) was spent in perids f spntaneus arusal (Table 1). Prtein synthesis Prtein synthetic rates were estimated in this study frm the incrpratin f L-[4-3H]-phenylalanine int pectralis muscle and liver prtein fllwing the intraperitneal injectin f a single flding dse. The validity f this methd depends n three Table 1. Duratin f trpr and arusal during hibernatin in E.fuscus Status Mean Minimum Maximum Ttal % duratin duratin duratin time ttal (h) (h) (h) (h) time Trpr , _+9.0" (86) ArusaI _0.2 (86) Mean 4-S.E. The numbers in parentheses dente sample size k U t- 0) s 0) 0) U_.>_ < O O. CO 800[ A. 6' ~}= = ~ ~ ~ ~ I - - i 400 l ~ 200[ summer looo OL--f/, 10 2'0 3'0 200 winler O -"/ ' 10 2O Time After Injectin (min] Fig. 2. The specific activity f plasma and intracellular free phenylalanine fllwing intraperitneal injectin with L-[4-3H] - phenylalanine in fasted summer bats and in hibernating bats arused frm trpr. Plasma (clsed circles) and liver (pen triangles) specific activities remain cnstant ver the 30 rain perid. Pectralis muscle (pen circles) specific activity decreases significantly (P<0.001) in summer bats, but remains cnstant in winter bats. Each pint represents the mean_+ SE f ~6 independent values O t33 >, O_ 0) 03 e-- r~ looo r 1 Time After Injectin (min) B. 3'0 12 Fig. 3. The specific activity f plasma and intracellular free phenylalanine fllwing intraperitneal injectin with L-[4-3H] - phenylalanine in trpid bats. Neither plasma (clsed circles), pectralis muscle (pen circles), nr liver (pen triangles) specific activity changes significantly between 10 and 120 rain fllwing injectin. Each pint represents the mean + SE f 4 independent values assumptins: (1) that the labelled amin acid equilibrates rapidly with the intracellular pl, (2) that the specific activity f the free amin acid in the intracellular pl remains cnstant ver the measurement perid, and (3) that the presence f a high intracellular cncentratin f phenylalanine des nt affect the rate f prtein synthesis. The validity f the third assmnptin has been demnstrated explicitly in rats by Garlick et al. (1980), wh shwed that the injectin f up t 150 gml

5 M.E. Yace: Prtein metablism in hibernating bats 141 Table 2. Prtein synthetic rate in pectralis muscle and liver f trpid and arused bats after 2 mnths f hibernatin and f fasted summer bats. All values are presented as mean_+ SE, the numbers in parentheses dente sample size Tissue Status Bdy Rate temperature f prtein (~ a synthesis (%/day) Pectralis muscle Summer 35.1 _+0.4 (17) 8.8_+2.2 (17) fasted P < b P < Hibernating, 33.5_+0.3 (15) 2.2+_1.6 (14) arused P< Hibernating, 3.5_+ 0.8 (8) < 1 trpid E c 0 Z) O_ 20 lo ~ L... 1() Time since last arusal (h) 260 3; Fig. 4. Tile cncentratin f urea in the plasma f E. fuscus during trpr (pen circles) and spntaneus arusal (clsed circles) Liver Summer, 35.1 _+0.4 Ct7) 63.0_+27.2 (12) fasted P< N.S. Hibernating, 33.5 _+ 0.3 (15) 53.3 i (15) arused P < Hibernating, (8) < 1 trpid a Final rectal temperature b The prbability that adjacent means differ due t chance, using a ne-tailed Student's t-test E f phenylalanine/100 g f bdy mass des nt affect the rate f prtein synthesis in either muscle r liver. Regarding the first tw assumptins, plasma specific activity rse rapidly fllwing intraperitneal injectin f 3H-phenylalanine, reaching a plateau value in less than 10 rain in all cases (Figs. 2, 3). Thereafter, plasma specific activity did nt change significantly in any f the grups. Similarly, the specific activity f free phenylalanine in pectralis muscle and liver samples als rse t a plateau value in less than 10 min, indicating rapid labelling f the intracellular free phenylalanine pl. Subsequently, specific activity did nt vary significantly during the measurement perid in any case except that f the pectralis muscle f summer bats (Figs. 2, 3). The specific activity f free phenylalanine in the pectralis muscle f summer bats underwent a slight, but statistically significant (P < 0.001) decrease between 10 and 30 rain pst injectin. Hwever, this decrease is small enugh that it des nt significantly affect the estimate f pectralis muscle prtein synthesis. Therefre, it appears that the assumptins f the technique used here are valid. The estimated rates f prtein synthesis in the pectralis muscles and liver f E. fuscus are presented in Table 2. The rate f pectralis muscle prtein synthesis in summer bats after an vernight fast is significantly greater (P<0.001) than that f hibernating bats arused frm trpr. On the c O O 8 O0 O0 0 Time since last arusal (h) Fig. 5. The accumulatin f urinary urea during trpr in E. fblschs ther hand, the rate f hepatic prtein synthesis des nt differ between fasted summer bats and hibernating bats arused frm trpr. The rates f prtein synthesis in bth the pectralis muscle and liver f trpid bats were belw the limit f detectin f the technique used (apprximately 1%/day). Plasma and urinary urea accumulatin in hibernating E. fuscus Plasma urea cncentratins are relatively high and variable in trpid bats ( ram), but they d nt vary with the duratin f trpr (Fig. 4). Ttal urinary urea increases significantly during trpr buts f up t 300 h (Fig. 5). This trend is described by the equatin: N= h where N is ttal urinary urea in I.tmles and h is the time since last arusal in hurs; r=0.78, P< 0.001, n=16.

6 142 M.E. Yace: Prtein metablism in hibernating bats Discussin During buts f trpr (bdy temperature= ~ the rates f prtein synthesis in the pectralis muscle and liver f E. fuscus are t lw t be detected with the methd emplyed in the present study. That is, n increase in the specifi c activity f prtein-bund phenylalanine was detectable tw hurs after injectin, despite rapid labelling f the free phenylalanine pls (Fig. 3, Table 2). On the ther hand, significant rates f prtein synthesis are evident in bth the pectralis muscle and liver f E. fuscus during perids f arusal (bdy temperature=33.5_+0.3 ~ after 2 mnths f hibernatin (Table 2). The rate f prtein synthesis in pectralis muscles f arused bats is significantly lwer (P<0.001) than that f summer bats fllwing an vernight fast. The rate f hepatic prtein synthesis in arused bats des nt differ significantly frm that f summer bats (Table 2). At the utset f this study it was expected that the rates f prtein synthesis wuld be very lw in bth the trpid and arused states in cmparisn with thse f fasted summer bats. In the case f trpid bats, lw synthetic rates were expected largely because f lw bdy temperatures. The bserved rates in trpid bats (Table 2) are cnsistent with this hypthesis. Hwever, the extent f inhibitin f prtein synthesis apparent in the livers f trpid bats (Q10 > 3.5) suggests that this inhibitin may invlve mre than a simple temperature effect. In the case f bats arused after 2 mnths f hibernatin, it was expected that prtein synthetic rates wuld als be lw as a result f time-dependent changes in prtein metablism similar t thse f nnhibernatrs during prlnged fasting. In nnhibernating mammals prtein synthetic rates underg an initial decrease during the transitin between the fed and pstabsrptive states (Millward and Waterlw 1978; Li etal. 1979; McNurlan et al. 1979) and then a further gradual decline which may result frm decreased levels f mrna (Millward and Waterlw 1978; Li et al. 1979) and decreased degradative rates which lead t reduced availability f amin acids (Gan and Jeffay 1967; Cahill 1976). The hypthesis that prtein metablism might underg changes during hibernatin similar t thse which have been bserved in prlnged fasting in nnhibernatrs was tested by cmparing bats arused after 2 mnths f hibernatin with summer bats after an vernight fast. These tw grups f animals had similar bdy temperatures (Table 2) and bth grups were in a fasted state. Hwever, the summer bats had fasted fr apprximately 24 h and the hibernating bats had nt had access t fd fr apprximately 2 mnths. The results (Table 2) d nt fully reslve this questin since the rates f prtein synthesis in the pectratis muscles f arused bats are significantly lwer than thse f fasted summer bats, but the rates f hepatic prtein synthesis d nt differ between these grups (Table 2). The high rates f hepatic prtein synthesis bserved in bats arused frm hibernatin cntrast sharply with earlier studies f hibernating grund squirrels (Spermphilus tridecemlineatus), which suggest that hepatic plyribsmes are deaggregated (Whitten et al. 1970) and that cell-free preparatins f livers have significantly lwer prtein synthetic capacities than d thse f summer squirrels (Whitten and Klain 1968). Since the hibernating bats used in this study had had n access t fd fr apprximately 2 mnths, the amin acids used fr prtein synthesis must have cme frm the breakdwn f existing prteins (see Gan and Jeffay 1967). Therefre, rates f prtein degradatin in hibernating bats shuld be equal t r greater than synthetic rates and the amunt by which the degradative rate exceeds the synthetic rate shuld equal the rates f net prtein lss. This simple relatinship is used here t estimate rates f prtein degradatin in trpid and arused hibernating bats and in fasted summer bats. The mst straightfrward case is that f the trpid bats. The rate f net prtein degradatin during trpr can be estimated directly frm the rate f appearance f urea. Urea des nt accumulate in the bld f bats during buts f trpr lasting up t 300 h (Fig. 4). Neither des plasma urea differ between the trpid and arused states (Fig. 4). Urea des accumulate in the bladder during trpr and is vided during peridic arusals (Fig. 5). The rate f accumulatin is very lw and crrespnds t a rate f lss f ttal bdy prtein f less than 0.01%/day. This amunts t nly abut 5% f the verall decrease in ttal bdy prtein which ccurs during hibernatin, despite the fact that trpr accunts fr 98% f the time f hibernatin (Yace 1982; Table 1). Because f the very lw rate f net prtein lss, the rates f prtein degradatin in pectralis muscle and liver must be apprximately equal t thse f prtein synthesis and therefre were assigned maximal values f 1%/day [this value crrespnds t the upper limit placed n the estimated rates f prtein synthesis (Tables 2, 4)]. The rate f lss f tissue prtein during peridic ar-asms was calculated as the difference between the lss f prtein mass in the pectralis muscle and liver during hibernatin (Table 3) and the amunt which culd be ac-

7 M.E. Yace: Prtein metablism in hibernating bats 143 Table 3. Changes in mass and prtein cntent f the pectralis muscle" and liver b in E. fuscus during 110 days f hibernatin Status Pec- Ttal Liver Ttal tralis pec- mass liver muscle tralis (g) prtein mass prtein (g) (g) (g) Pre _ _ _ _+0.02 hibernatin (n = 11) (n = 11) (n = 1 i) (n = 11) fattening P<0.001 ~ P<0.001 P<0.001 P<0.001 After 0.88_ _ _ _ days f (n = 14) (n = 14) (n = 14) (n = 14) hibernatin " Values taken frm Table i, Yace (1983) b Unpublished data cllected frm the same grup f animals frm which the muscle data were cllected. Methds were identical t thse described in Yace (1983) fr the determinatin f pectralis muscle mass and prtein mass c Prbability that adjacent means differ due t chance, using a ne-tailed Student's t-test Table 4. Estimated rates f net lss f prtein mass and prtein degradatin in the pectralis muscle and liver f E. fuseus. Calculated frm data in Tables 1-3 and Figs. 5 and 6, as described in the Materials and methds sectin Status Tissue Rate (%/day) Net prtein lss Degradatin Summer, Pectralis > 0 > 9 fasted muscle Liver > 0 > 63 Hibernating, Pectralis arused muscle Liver Hibernating, Pectralis 0.01 < 1 trpid muscle Liver 0.01 < 1 cunted fr by urea prductin during trpr. Assuming that apprximately 95% f the net prtein lss ccurs during arusals and that arusals accunt fr 2% f the verall time in hibernatin (Yace 1982; Table 1), these calculatins yield estimates f 20%/day and 23%/day fr the fractinal rate f lss f pectralis muscle and liver prtein, respectively, during peridic arusals. Therefre, the fractinal breakdwn rates fr pectratis muscle and liver prtein during peridic arusals are estimated t be 22%/day and 73%/day, respectively (Table 4). Using similar reasning, if the summer bats were in a steady state with respect t tissue prtein mass, the fractinal degradative rates wuld equal the fractinal synthetic rates (8.8%/day and 63%/day fr pectralis muscle and liver, respectively). Hwever, after an vernight fast the bats were undubtedly in negative nitrgen balance, s these estimates represent lwer limits n the degradative rates in muscle and liver f fasted summer bats. Despite the crude nature f these estimates, the data suggest that the rates f prtein breakdwn in bth pectralis muscle and liver during peridic arusals are similar t the crrespnding rates in summer bats. These data fail t supprt the hypthesis that prtein metablism underges changes during hibernatin similar t thse in prlnged fasting (see Felig et al. 1969; Yung et al. 1973; Gldberg and St. Jhn 1976; Felig 1979; and Le Mar et al. 1981). Rather, the high rates f prtein breakdwn and net prtein degradatin in bats arused frm hibernatin are cmparable t thse seen in shrt term fasting, when tissue prtein is rapidly degraded t supply the increased glucnegenic demand (Adibi 1976; Pzefsky et al. 1976; Chang and Gldberg 1978a, b; Li et al. 1979; Snell 1980). Glucse xidatin appears t accunt fr a significantly greater prprtin f metablism during peridic arusals than during trpr buts in hibernating mammals (Suth and Huse 1967; Tashima et al. 1970; Yace 1982). Since glycgen stres are small (Ddgen and Bld 1956; Lenard and Wimsatt 1959; Yace 1982) and the cntributin f glycerl is cupled t the rate f fat xidatin, the increased demand fr glucse might be met by glucnegenesis frm tissue prtein. The results presented here, indicating relatively high rates f net lss f pectralis muscle and liver prtein, tgether with the earlier bservatins that hepatic glucnegenic capacity increases during hibernatin (Klain and Whitten 1968) and that urea is prduced in large quantities during spntaneus arusal (Galster and Mrrisn 1975), suggest that spntaneus arusal is a perid when tissue prtein is degraded t supply glucnegenic demand. On the ther hand, the very lw rates f tissue prtein synthesis and degradatin bserved in trpid bats suggest that prtein metablism is virtually suspended during trpr. Acknwledgements. I wuld like t thank W.R. Dawsn fr his help thrughut this prject. This study represents a prtin f a dissertatin submitted in partial fulfillment f the requirements fr the PhD degree. This study was supprted in part by a blck grant frm the Hrace H. Rackham Schl f Graduate Studies, The University f Michigan.

8 144 M.E. Yace: Prtein metablism in hibernating bats References Adibi SA (1976) Metablism f branched-chain amin acids in altered nutritin. Metablism 25: Beer JR (1955) Survival and mvements f banded big brwn bats. J Mammal 36: Beer JR, Richards AG (i956) Hibernatin f the big brwn bat. J Mammal 37:31M1 Cahill GF Jr (1976) Starvatin in man. Clin Endcrinl Metab 5 : 397M15 Chang TW, Gldberg AL (1978a) The rigin f alanine prduced in skeletal muscle. J Bil Chem 253 : Chang TW, Gldberg AL (1978 b) The metablic fates f amin acids and the frmatin f glutamine in skeletal muscle. J Bil Chem 253 : Ddgen CL, Bld FR (1956) Energy surces in the bat. Am J Physil 187: Felig P (1979) Starvatin. In: DeGrt LJ (ed) Endcrinlgy, vl 3. Grune and Strattn, New Yrk, pp Felig P, Owen OE, Wahren J, Cahill GF Jr (1969) Amin acid metablism during prlnged starvatin. J Clin Invest 48 : Fricke U (1975) Tritsl: A new scintillatin ccktail based n Tritn X-100. Anal Bichem 63 : Galster WA, Mrrisn PR (1975) Gtucnegenesis in arctic grund squirrels between perids f hibernatin. Am J Physil 228: Galster WA, Mrrisn PR (1976) Seasnal changes in bdy cmpsitin f the arctic grund squirrel, Citellus undulatus. Can J Zl 54:74-78 Gan JC, Jeffay H (1967) Origins and metablism f the intracellular amin acid pls in rat liver and muscle. Bichim Biphys Acta 148: Garlick PJ, McNurlan MA, Preedy VR (1980) A rapid and cnvenient technique fr measuring the rate f prtein synthesis in tissues by injectin f [3HI phenylalanine. Bichem J 192: Gldberg AL, St Jhn AC (1976) Intracellular prtein degradatin in mammalian and bacterial cells: Part 2. Annu Rev Bichem 45:74%803 Gdman MN, Ruderman NB (1980) Starvatin in the rat. I. Effect f age and besity n rgan weights, RNA, DNA, and prtein. Am J Physil 239 : E269-E276 Klain GJ, Whitten BK (1968) Carbn dixide fixatin during hibernatin and arusal frm hibernatin. Cmp Bichem Physil 25 : Le Mar Y, Van Kha HV, Kubi H, Dewasmes G, Girard J, Ferre P, Cagnard M (1981) Bdy cmpsitin, energy expenditure, and plasma metablites in lng-term fasting geese. Am J Physil 241 :E342-E354 Lenard SL, Wimsatt WA (1959) Phsphrylase and glycgen levels in skeletal muscle and liver f hibernating and unhibernating bats. Am J Physil I97: i Li JB, Higgins JE, Jeffersn LS (1979) Changes in prtein turnver in skeletal nmscle in respnse t fasting. Am J Physil 236 : E222-E228 McNurlan MA, Tmkins AM, Garlick PJ (1979) The effect f starvatin n the rate f prtein synthesis in rat liver and small intestine. Bichem J 178: Millward D J, Waterlw JC (1978) Effect f nutritin n prtein turnver in skeletal muscle. Fed Prc 37: Pzefsky T, Tancredi RG, Mxley RT, Dupre J, Tbin JD (1976) Effects f brief starvatin n muscle amin acid metablism in nnbese man. J Clin Invest 57: Snell K (1980) Muscle alanine synthesis and hepatic glucnegenesis. Bichem Sc Trans 8 : Suth FE, Huse WA (1967) Energy metablism in hibernatin. In: Fisher KC, Dawe AR, Lyman CP, Schnbaum E, Suth FE (eds) Mammalian hibernatin III. Oliver and Byd, Edinburg, pp Suzuki O, Yagi K (1976) A flurmetric assay fr fl-phenylethylamine in rat brain. Anal Bichem 75: Tashima LS, Adelstein S J, Lyman CP (1970) Radiglucse utilizatin by active, hibernating, and arusing grund squirrels. Am J Physil 218: Wang LCH (1978) Energetic and field aspects f mammalian trpr: The Richardsn's grund squirrel. In: Wang LCH, Hudsn JW (eds) Strategies in cld: natural trpidity and thermgenesis. Academic Press, New Yrk, pp Whitten BK, Klain GJ (1968) Prtein metablism in hepatic tissue f hibernating and arusing grund squirrels. Am J Physil 214: Whitten BK, Schrader LE, Hustn RL, Hnld GR (1970) Hepatic plyribsmes and prtein synthesis: Seasnal changes in a hibernatr. Int J Bichem 1:40(~408 Yace ME (1982) The maintenance f pectralis muscle during hibernatin in the big brwn bat, Eptesicus fuscus. PhD thesis, University f Michigan, Ann Arbr Yace ME (1983) Maintenance f the pectralis muscle during hibernatin in the big brwn bat, Eptesicusfuscus. J. Cmp Physil Yung VR, Haverberg LN, Bilmazes C, Munr HN (1973) Ptential use f 3-methylhistidine excretin as an index f prgressive reductin in muscle prtein catablism during starvatin. Metablism 22: