Protein metabolism and urea recycling1n

M. L.

hibernators'

DepaltmtnL oJ'Biolou, Unit,ersitj of'1,{tiu Mcxico, Albuqrtcrque, i{eiu Mexico B7 1}1

rodentRIEDESEL AND J. M. STEFFEN

L,1,.,."nt in thc sizc of'roclcnts anclolhcl srnall rnarnnr:rls is a lelativell,high rnetabolic rate ancl a linritcclcapar:itl' firr eneruy stor:lgc. In adcli-tion the lalgc surface al'ea to r,olunreratio results in elevalecl r:rtcs ol'hcatloss firllorvinq exposur-c to l()rv en-r.'ir<ltrnre ntal temperaturcs. The meta-lrolit: dernancls irnposecl bv srnall l;ocllsize alc arnplified n'lien pcriocls oi'lccluccd fbocl and fi'ee rvater av:ril-:rbilitl' coincicle lr'ith seasonallv krrv-crccl ambient temper:rtLrles. Hibcrrra-tion rcpresents a biolosical aclapta-fion that le:rclily meets thc energ)'cletrurncls of small animals in colclclinrutes.

Wanu (30) has recenf.lv evaluatecltltc cnelgetics firr seasonal torpol ofSpermophilus richtLrdsonil in the fielcl.These clata agree clrrite favorably rvithKayscr's (16) classic revierv of theclirt:r c:ollected on r::rptivc animals. ltis apparent fronr these stuclies th:rtI at'ge nr:rm tnalian h iller'n:rtors, suclt :rsthe bear, irre in a vel'y cliff-erentenergy balance situation than r.trdenthiberrrators. Bears I'rave typicallybcerr firuncl to lose l5 to 257o of thcirbocly r.veiuht ovcr thc period of'con-tinuous r,vinter hibernation rvhereasrcrdents nray lose 40 tc> 50% of thebocly wcieht. Ninety' percent of tireerlersv utilizecl during the hiberna-tion seasrrrr <tf S. rir:ho,rdsonil is asso-ciatecl rvith the 30 C elevation ofbodl' temperature experienced dur-ing perioclic alousals ancl the follorv-ine phase of normothermia (30).

Hibernators have devektpecl markedacl:rptations to both recluced bodyt.emperature and prokrngecl Ihsting.Northern species may spencl up to 8or 9 months in hibenration (23) rvithlittle or no food intake (13). Energyreserves are not consiclered to be alimiting factor in rodent hibernation.In fact rnany rodents have been ob-

0014-9446/80/0039-2959/$01.25. O FASEB

Riedesel, M. L., and J. M. Steffen. 1980.urea recycling in rodent hibernators.2959-2963. Invited paper.

Protein metabolism andFederation Proc. 39:

ABSTRACT

A rt:r'iel of the nretabolism of hibernators supports the argument that proteitr utilization is

:rrr cssenti:rl aspect ol hibelnation. Ser,cral lines of'evidetr<:e in support of this hvJrothesis arerlist:Lrsserl, inclucling (1) the ficld prefererrt:es of some hibernators lor a rliet rich in protcirrprior lo hibernation, 12) thc glur:oncopenic rnainlenance ol gll'cogcn stores duringhibenration, (i) thc nraintcnarrr:e ol pl:rsrna amino acicl ler,cls drrrinc hibelnation and theirelcr,ation drrring the l)rocess ol arousal, (4) changes irr lcvcls of nonllrotein nitrogen lvithhibelnation, (5) the potential for urea rccl'cling bv intestinirl rnicloflora in active fastecl

aninrals, lruci (6) thc lack of'corrclalion bctrveen Ioss ol lean bodv rnass ancl ure:r recoveryclrrring tlre hiberrn:rting season. Thcsc d:rta scrvc to enrphasize the contribution of proteinrnctalrolism to the o\er:rll encrgetic acliustlltcnts re<lrrired fbr sulvival at a lorver bodytcnrpclrtule.-Riedesel, M. L.; Steffen,J. M. Prot.ein niet:rbolism and urea reclcling inroclcnt lriberrralors. Federatian Prot. i\l): 2959-2963: 1980.

scrved not to eat for sever:rl u,eeksfollorvins the ternrinal irrotrsal fromhibcrnation in the sprinn. Despitethe eyidence of arlple enel.gy rcservcsr,I r)r:rlrrrrraliarr lrihernltols, irrresli-sal.ors tencl to emphasize stored lipiclenergv as thc kel'tiutriti<lnal factot're-sponsible firr the sucr:ess o{ hi}ret'nation.

I rr :rr r intn I n r rt rit ir,n. proleit t is

rer:oqnizccl as being essential f orstructural, enzvmatic, irnd osmoticfunctions. Certzrinly these basic re -

quirements must bc nrct in the hiber-nating :lnimal. .|ust ils there areclifferer-rt strategies in meeting thebasic neecl fbr protein among anirn:ilsirr general, it is to bc cxpected thatthere :rre also clill'crcrlt strateqies foraccluisition, utilization, and conserva-tion of protein among hibelnators.The ,rhjct tire ,,f this plescrrtatiort is

to summarize evidence that iclcntifiesadaptive acliustments in protein me-tabolism that are as essential to thehibernating nrdent as is the accumula-tion of'body fat prior to hibernationand oxidation of lipicl as an cnersysoulce d rrring lrihcln:rl ion.

DIET AND BODY COMPOSITION

Fielcl observation, inclucling the ex-arnination of' stomach c()ntents. dc-

scribes a distin<:t preference in thesolden-mantlecl ground squirrel andseveral chipmunk species for a diethigh in protein (22, 28, 29). 'fhispref erence is cspecially apparentclurinq thc 1-:rll prior to hibernation.The clevated level of lipid de positionin the fall coincides rvith a diet offungi high in protcin. 'l'he recentdemonstration by Montoya et al. (24)that protein deprivzrtion in the gardendt,rm,rusc indur ed t()rpol irr Jn)season emphasizcs the f'act thatpr<itein may bc a limitins factor inthe metabolism ol hibernators. Bothlipirl and protein lcserves may in-f luence the onset, duration, andsuccess of hibernation.

Changes re ported in lean bodymrtss irrdit ate thal prorcin nlay a( -

count for approximately l0%: of thecnetgy expenditure during the hiber-natins season ( 12) Jame son andMead ( 14) noted conservation offat-free dry rveieht both in animalshoardins loocl for consumption dur-ing periodic arousals, as do chip-

1 Fronr tlre Symposium Protein and FatMetabolism during Mammalian Hlpophag'ta andLlibernation presented at the 30th Annual FallMeeting of The American Physiological So-r:iety, New Orleans, October 19, 1979.

2959

munks, and in those that do not ezrt,

such as ground squirrels. Holr,et,er,even though chipmunk spccies cachefood prior to hibernation, theyundergo prehibe rnation fatteninsthat is as extensivc as the fatteningin the ground squirrel (14) Thecachir-rg of food reserves may resultprimarily from the demand {brprotein rather than the neecl forenergy. Although the storage of f'at as

an energy sourcc f or utilizationduring hibernation is well-establishedin ground squirrels, there are n<r

studies that examine the minimumbody protein recluired prior to hiber-nation. Carpenter (7) has reportedthat the declinc in nitrosetr elirnina-tion cluring hibernation paralleled thedecline in oxygen c()nsumption. Ca-tabolism of protein when thc renalrorrle of ttitrogett ex( rcl ion is c\\ett-tially nonluncli,rrtal mav nt t estit:tl<'the stclrage or recvcling cif nitrogerr.

GLYCOGEN RESERVES-l'he maintenance of ulvcogen st()resin both muscle and liver throughouthibernation serves as zr clue to theirnportance of gluconeogenesis dur-ing hibernzrtion (16, 34). -fhc inter-pretation of data collected on animalshibernatirrg irr the lab<,ralory is r,rnt-plicated by the recent report o1'Bintzet al. (5) on fielcl anirnals. 'I'he fielcl-killed animals had live r glycoecnvalues less than 8% oI' labolatoryacclimated anirnals. Grouncl squirrelshoused in the laboralory fbr I yeararrcl f:rsted firr 8 days to 30% rveipJhtloss had 4.5 times the liver gl,vcoecn<lbserved in the field anirnals. Bintz'sdata support the hypothesis thzrtgluconeogenesis may be a rnoreimportant source of blood glucosethan is glycrigenolysis firr the fielclanimuls. Hrrrrevet thcrt' is tl() ( ()ll-

Kristoffersson and Broberg (19) re-ported lor'r,ered plasrna levels for rnostamino acids during hibernation of the

2960

nonrodent heclgehog, although as theperiod of hiberrration r'vas extendeclthese values increased slightly. Plzrsnralevels of' valine, leucine, isoleucine,lvsine, and ornithine \{ere elevateclthtrughout hibernation.'l'his samestudy reported no changes in plasmaarnino acid conce nttatiolr in thehamster cluring hibernati<irt. Klair-rirrrd Whitten (17) exanrined S. tri-rlecemlirteatus and reported little changein plasrna :rmino acid concentrationsrvith hiberr.ration. Plasma lcucine,:rreinine, and alanine rvere the ortlyamino acid concer)tr:ltions that in-tle:rscd u ith lribelrr:rtit,n.

Arnino acids have been recognizedas an irnportant s()rlrce ol e ne reydurins arousal frclm hibernation;this is inf'errecl trorn both increasedlevels of plasnra amino acids (18) andclevated rates ()f':rmino acid oxiclationclurins the arousal pr(xress (6, 3l, 32).Alanine has irnportant roles in bothnitrogen rnetabolism and gluconeo-senesis. In the glucose-alanine cyclc,skelet.al muscle scl-ves as the sourcco{'alanine. Following uptake o{' thisarnino acicl by the liver, the anrinogroup may be {urrrreled to either urcaproduction ()r synthesis ol otheramino acicls. The carbotr skelettin maysubsecluently be c<invertecl to glucosc.Plasma lcvels clf'alanine rise markedlycluring arous:rl of the l3-lined sroundsquirrcl (17), as cloes the rate ofalanine conversion to glycogen (32).As mentioned earlier, the cost ofarousal ancl periodic norrnothermiaaccounts lirr 90% of the e ncrsyexpenditure during the winter hiber-natins se:lson (30); thus alanine anclother amino acids nray represent a

large portiotr ofthc cnergy expendi-Irrle rturirtrr thc Iribernating seasort.

NONPROTEIN NITROGEN

several-fbld higher in hibernatorsthan in mammals in seneral (10, 25).With re spect to pl:rsma ()r serurrr urea

concentrations, horvever, there isccinsiclerable contrr)versy in the lirnitedliterature. Plasma ure a concerltra-ti<ins have been reporte d not tochange durins hibernation in S.

tridecemLineatus (8), S. coLutnbianus(20), and Malmotu, .flaaiuentri.s (15).However, Passrnore et al. (27) rep()rtincreased plasma urea in S. columbi-nnzs while Fisher (10) and Galsterand M<lrrison (l l) have noted reduc-tions in S. Lateralis and S. undulcttus,respectively. Obviously there is noconsistent pattern that may be de-duced from these clata on urea.

Analysis of tissue content of non-protein nitrogen has resulted ir.r

several interesting observatiot-ts. Kris-tofferson (18) rep<lrts an elevatedurea content of both diaphragm andsmall intestine durins hibernationin the hedeel'rog. In addition, Err-rir-bekov and Mukailov (9) report in-creasing arnmonia c()ntent in bothcerebrum and celebellum with in-creasing duration of hibernation inS. pygmacus. At the same time thatamrnonia content increased, ureaconcerltration fell.

The recent demonstration thatmethylated arnino acids n'ray serve as

an index <ll skeletal muscle proteintLrl'nover shoulcl be applied to hiber-nators (33). Deterrnination of theamount of rnethylated amino acidsaccumulated during hibernaticxr wouldidentify the extent of uluconeogene-sis fiom skeletal muscle proteins.Data we have collected on summeranirnals indicate urine- and blood-

TABLE 1. Calculated water balance ina rodent hibernator

Sourus oJ'uaterFrec water (adipose - 25%; muscle - 75%)

Adipose, l0 kcal : I . 1 g yields 0.4 ml rvaterMuscle, 10 kcal : 2.5 g yields 7.5 ml water

Metaboli<: u'ater (fat : 1.07 ml/g; muscle

- 0.4 rnl/g)Aclipose, 10kcal - 1.1 eyields 1.2 ml waterMuscle, 10 kcal : 2.5 g yields i.0 ml water

Iter uatt-r pius meltb()lic watet'Fat - O.4 + 1.2 - l-6 rnlProtein - 7.5 + 1.0 - 8.5 ml

Eu't!,'rnlit t turtlcr 1,"' I EWLtOxygen neecled EWL

per 10 kcal 0.70 g/liter O,Fat - 2.2 liter Fat : 1.54 gProtein = 2.4 liter Protein - I.68 g

Gain less lo.ss

Fat - 1.6 - I.54 - 0.06 gainProtein : 8.5 * 1.ti8 : 6.8 gain

clusive evidence regarding the exte-nt ]'he hypethesis that accunrulati.nto rvhich gluconeogenesis in the 6f' nitr',rgep()1s \\astes and the re-hibernating rodent rcsults f rtlm trti- sultant to*i.iry alcl osrn.tic intbalancelization of'- anrino acicls <ir glycerol are cagsal factgrs ip the perioclicderived frorn the breakdown oi' altusals {}rm hibernatiol has re-neutral fats. ceived attention for a number of

years. The evidcnce that renal func-

AMINO ACIDS tion i.s inrpaired during hibernation(27) lent inclirect support f or the

Plasma concentration ol amin<i acids hypclthesis, as did early measure-may serve as a useful indirect Ineasure ments OI'plasrna nonprotein nitrogenof the rate of proteiu tul'Ir()vcl'. concentrati<tns, lvhich revealed values

FEDERATION PROCEEDINGS VOL.39, NO. 12 OCTOBER 1980

o=

o

urea values are n()t g(x)d indicat()rsof'the extent of protein catabolism.

MUSCLE AS A SOURCE OF WATER

Whereas hiberrrati,rrr is rrr energt'ti-cally successf ul approach to cr()nservii-

Figure 2. 'I-irrre course r>f bloorl rrrea charrqesfirlkrn'ing urea loading. Ve rtical lines lepreserrt

tion of limited resources, the state ofhibernation is also ass()ciated lvith anumber of limitations clr disadvan-tages. Of paramount concern is theextended absence o{' water intake inthe face ofa reduced, but continuous,evaporative water loss (EWL). 'I'heEWL is minimized in part by thehibernating posture, which promotesrebreathing of humidified air (4).Loss of' body water, much of'n,hichmay be ascribed to pulnrontrry in-sensible water loss, arn()Llrlts t() onequarter of the total weight krss cluringthe hibernation season (12). It rvouldbe of advantage, therefitre, to con-sider not only the energy availablef rom catabolism of a specific tissuebut also the potential f<rr procluctionof'rnctabolic water or release Of' freewater. In contrast to its relativcabsence in stored lipid, free waterrepresents 7 5% oI the mass of muscletissue. As a result of' the elevatedventilation required to provide zrcle-quate oxygen fbr complete oxidatirtnof highly reduced lipid compounds,the total water available {'rom oxida-tion clf' fat is near the pulrnonaryF,Wl- value. Catabolisnr of protein

yields more water per calorieenergy than does catabolismfat (Table l).

UREA CATABOLISM ANDNITROGEN RECYCLING

Recycling of urea nitrogen in a largehibernator, the black bear, has beendescribed by Dr. Nelson in thissymposium (26) and recycling mightwell be anticipated to take on a some-what different form in smaller speciescapable of hibernation. There arernajor differences in both physio-logical and metabolic aspects ofhibernation between bears and smallermammalian hibernators; for instance,capacity for energy storage, depth oftorpor, and periodic arousals insmall hibernators.

'I'he potential firr urea recyclinghas been studied in fasted and infasted, water-deprived (FWD) groundsquirrels (5. LateraLis). These condi-tions, lvhich are accompanied by a

reduction in urine f<rrmation andexcreti()n, mimic the situatic-rn facedannually during hibernation. Bintzet al. (2, 3) describe a pre{'erencefcrr protein catabolisrn in the food-and water-deprived golden-mantledground squirrel. In terms of' waterbalance tl're advantage of protein overfat catabolism is evident from theabove discussion. The 60 to 70%reduction in urea excretion associatedwith fasting or FWD in S, Lateralis(Fig. l) appears to be in conflict withBintz's evidence of increased proteincatabolism under the same conditions.

Urea-loading experiments involv-ing laboratory rats and ground

Figure 3. Tinrt tourrc ol urine urer exctetion

l:11:"'"t urea loading. Vertical lines represent

Qcrcuuo sutRl€Ls

l) tae nars

ofof

600

500

3 4ooFUJe,

I 3oo

UJ

fi 2ooE,f

100

DAYSFigure l. Seasonal effects of f astins or Iirstins:rnd warer deprivation (FWD) on daily urea excre-tion. FWD rats (Dec.) a

- a FWD p;round scluirrels (May) n

- [l F-WD ground scluirrels

(Dec.) I -

l fiisted ground squilrels (May-July) O

- O. Vertical lines represenr seivr.

I3

!oo

a

5Uzrlz

UEl)F

P

so5flEloaJm

DISAPPEMANCE OF UREA INJECTION

PROTEIN AND FAT METABOLISM DURING MAMMALIAN HYPOPHAGIA AND HIBERNATION 2961

squirrels injected intraperitoneallywith 0.3 g urea per 100 g body weightreveal an absence of plasma ureaaccumulation in the ground squirrel(Fig. 2). The same load results in a

threefbld elevation of plasma ureain the rat. The absence of plasmaurea accumulation is not a result ofmore efficient renal excretion of ureain the ground squirrel, as evidencedby the fact that urea excretion ismaintained at or below the levelsseen in the rat (Fig. 3). We may con-clude from this evidence that a ureaload sufficient to elevate blood ureathreefold in the laboratory rat doesnot cause short-term accumulation ofurea in the blood or enhance ureaexcretion in the ground squirrel.

Catabolism of [laC]urea has alscr

been examined in FWD S. lateralis. lnthese animals, fasting for'4 days cclnr-bined with 24 h of water deprivationresults in a lO-fold increase in uleacatabolism and a 30-fold reductionof labeled excretion (Table 2). Severalmonogastric mammalian species, in-cluding man, have been shown toharbor microorganisms capable ofurease activity (21). Treatment ofFWD ground squirrels with severalmicroflora-reducing antibiotics re-sults in return of urea catabolismto near control levels. In addition,antibiotic treatme nt results in en-hanced excretion of urea, with drug-treated squirrels excreting three timesas much urea as fasted animals, thusindicating storage or recycling ofapproximately 75% of the ureaproduced in fasted animals (Fig. a).These data suggest that retention andcatabolism of urea may be com-ponents of the metabolic response tofood and water deprivation in theground squirrel.

Although there have been no directstudies of' the potential for urearecycling in the hibernating state.examination of the literature revealsevidence of urea recycling. Both inthe hibernating arctic ground squirreland in the hibernating marmot, lossof lean body mass cannot be accounted

REFERENCES

I. Benedict, F. G,; Lee, R. C. Hibentation andma,rmot physiology. Carnegie Inst. Wash-ington Publ. 497, Baltimore: WaverleyPress; 1938, table 36.

2. Bintz, G. L. Oxidation of laC-labeled

carbohydrate, fatty acid, and amino acidas a cr-iterion of selective tissues catabo-

2962

TABLE 2. Percentage of inlected taO collected in urine and expired air in control,fasted-water-deprived, and fasted-water-deprived-drug treated ground squirrels

Percentage of injected taC

collected in 24 hT'wenty-lour-hoururine volume (ml) Urine CO, trap

Control (N : 9)Fasted-water-deprived (N : 8)

f penicillin (N : 5)+ sulfa (N : 4)* tetracycline (N : 3)

8.9 + 0.7o

1.1 * 0.7

79 + 4.42.6 + 0.80

2.4 + 0.I526.5 + 0.807.3 * 0.94.4 + I.21.4 + 0-6

u Mean I sr.

Figure 4. EfI'ect of lasting or f asting combined with drug treatment (FDT) on daily weight-specificurea excretion. Vertical lines represent sEM.

-c.9o)

=Eod)ctroIc'6!td,

EoxlrJ

E:)otr

=45

Doys of Fostirg

fbr by excreted urea (l, I l, 12). Thusthese animals exhibit charactelisticssimilar to fasted water-deprived activeS. lateralis in which the potential Ibrurea recycling was high.

In summary, the ability of thehibernatol to recycle urea nilrogen.utilize anrino acids as a major source

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FEDERATION PROCEEDINGS VOL.39. NO.12 OCTOBER 1980

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PROTEIN AND FAT METABOLISI\4 DURING MAMMALIAN HYPOPHAGIA AND HIBERNATION 2963