Proc. Natl. Acad. Sci. USAVol. 89, pp. 7119-7123, August 1992Biochemistry

Central role for differential gene expression inmammalian hibernation

(regulatory evolution/acute-phase response/a2-macroglobulin)

HILARY K. SRERE*, LAWRENCE C. H. WANGt, AND SANDRA L. MARTIN*t*University of Colorado Health Sciences Center, Department of Cellular and Structural Biology and Program in Molecular Biology, Box B111, 4200 EastNinth Avenue, Denver, CO 80262; and tDepartment of Zoology, University of Alberta, Edmonton, Alberta, Canada T6G 2E9

Communicated by Dale Kaiser, April 22, 1992

ABSTRACT Mammalian hibernators experience dramaticreductions in body temperature, metabolic rate, respiratoryrate, and heart rate during hibernation. These changes areprecisely controlled and reversible with only internally drivenmechanisms, suggesting specific biochemical regulation. Wepresent a model that integrates our observations of differentialliver gene expression during preparation for, and maintenanceof, the hibernating state, with the known phylogenetic inter-spersion of hibernating species in several major mammalianlineages. This model predicts a major role for the differentialexpression of existing mammalian genes in the biochemicalregulation of hibernation.

Hibernation is an adaptive strategy that is used by species inseveral mammalian orders to conserve energy in cold orinhospitable environments. During hibernation, these mam-mals dramatically lower their metabolic, heart, and respira-tory rates as well as their body temperature in a preciselycontrolled manner. This strategy helps some, particularlysmall, mammals to survive cold winter conditions in temper-ate climates when food and water are scarce yet the demandfor metabolic heat generation is high. Hibernating species arefrequently closely related to nonhibernating species and arefound in five orders of eutherian mammals as well as in a fewspecies of marsupials and monotremes (ref. 1 and Fig. 1). Theadaptive significance of hibernation, coupled with the inter-spersed phylogenetic distribution of hibernating speciesamong mammals, makes hibernation an ideal model systemto test the hypothesis that the molecular basis of importantadaptive events during evolution involves mechanisms thatlead to the differential regulation of existing genes and not theinvention of new genes (reviewed in ref. 3).The evolutionary origin of hibernation is unknown; either

of two hypotheses can explain the interspersed nature of thedistribution of extant hibernating species throughout Mam-malia. One postulates that the common ancestor of allmammals was a hibernator, but the ability to hibernate waslost independently along multiple lineages. The alternativeproposes that the common ancestor of all mammals was nota hibernator, and the ability to hibernate has been gainedindependently along multiple lineages (Fig. 1). With eitherscenario, we predict that a small number of regulatorychanges determine the phenotype of hibernation, rather thanthe de novo creation or loss ofnumerous hibernation-specificbiochemical functions, encoded by a collection of hiberna-tion-specific genes. Thus, hibernation must be the result of areprogramming of existing mammalian biochemical capabil-ities through the differential expression of existing genes.There is little evidence in the literature for differential gene

expression during hibernation, although there are a fewreports that demonstrate increases or decreases (4) in the

amount of specific proteins relative to the active state.Mitochondrial uncoupling protein (thermogenin) is an exam-ple of a protein that increases in amount dramatically duringhibernation. The mechanism for achieving this increase,however, is not differential expression of the gene encodingthermogenin. Instead, there is an increase in the amount ofbrown fat tissue in the animal and an increase in the numberofmitochondria per cell (5). Serum albumin is another proteinthat has been reported to increase during hibernation (6, 7).Serum albumin is produced by the liver, an organ that playsa primary role in the maintenance of internal homeostasis inall mammals. Such a role is likely to be critical during thephysiological extremes experienced during hibernation. Forthese reasons, we set out to test our hypothesis that differ-ential gene expression plays a central role in the adaptationof hibernation by looking at liver gene expression, beginningwith albumin.

MATERIALS AND METHODSAnimals. Richardson's ground squirrels (Spermophilus

richardsonii) were trapped (using National traps) in thevicinity of Edmonton, Alberta, Canada, from the time ofemergence (mid-March) until the time of immergence (lateSeptember to early October). Columbian ground squirrels(Spermophilus columbianus) were obtained on the easternwatershed of the Rocky Mountains southwest of Calgary,Alberta. Animals were housed in shoe box cages with foodand water ad libitum and were used within 1 year. Thoseanimals not sacrificed within 3 days were weighed weekly todetermine their state in the annual hibernation cycle; animalsshowing a rapid increase in body weight were placed in anenvironmental chamber at 30C in total darkness to facilitatehibernation. Animals showing a low body weight weredeemed to be in the nonhibernating or "active" phaseregardless of the time during the calendar year. No animalwas sacrificed as a hibernator unless it had previouslycompleted at least one hibernation bout [the hibernationseason is normally punctuated by periodic arousals to eu-thermy, leading to "bouts" of hibernation (1)] and was indeep hypothermia. Individual animals are considered here asbelonging to one of three groups: Group A animals (sixindividuals) were Richardson's ground squirrels that weresacrificed on the same day (season-independent); three ofthem were active and three were in deep hibernation (state-dependent). Group B animals were also Richardson's groundsquirrels, but plasma samples from active summer (earlyJune) and late fall (mid-October) animals were collectedwithin 3 days of capture from the wild (thus, these animals areseason- and state-dependent). The hibernators in this groupwere maintained in the laboratory as described above, but thedates of sacrifice varied. Group C animals were Columbianground squirrels; all four were sacrificed on the same day.None of the animals was studied serially.

tTo whom reprint requests should be addressed.

7119

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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commonancestor _is a

hibernator

primates

bats

insectivores

ungulates

carnivores

rodents

|agomorphs

marsupiats

monotremes

commonancestoris nct a

hibernator

FIG. 1. Two possible evolutionary histories for the adaptation of hibernation. The dendogram (based on the classification of Eisenberg, ref.2) shows the distribution of hibernators (1) among the major mammalian lineages. Lineages with no known hibernating species are shaded. Thetree on the left assumes that the common ancestor of all extant mammals was a hibernator, and shows the lineages that have completely lost,-, or partially lost (-) the ability to hibernate. The tree on the right has a nonhibernating ancestor, and thus the lineages where hibernationmust have arisen are marked (+). Within all of the lineages where hibernating species are found, species that hibernate are interspersed withspecies that do not. Thus multiple, independent gain and/or loss events are required to explain the present distribution of hibernating speciesin these lineages. The classic "deep hibernators," where body temperature may fall below 0C (30) are found among rodents, insectivores, andbats (1).

Biochemical Assays. The amount of albumin was deter-mined by an automated bromcresol green binding assay (8)and the total protein was determined by either an automatedbiuret (9) or a Bradford (Bio-Rad) assay. a2-Macroglobulinactivity was measured by the Ganrot assay (10). Sampleswere analyzed within the linear range of each assay.

Protein Purification and Antibody Production and Use. Fiftymicroliters ofplasmafrom a hibernating Richardson's groundsquirrel was diluted to 0.5 ml with water. Solid ammoniumsulfate (0.1472 g) was added with stirring at 40C for 20 mmn.The precipitate was collected by centrifugation at 16,000 x gand suspended in sample buffer for SDS/PAGE (7.5% acryl-amide, ref. 11). The protein was recovered by electroelutionand injected into rabbits with Freund's complete adjuvant(12). The resulting antiserum was used on Western blots (31)and plaque lifts at a 1:1000 or 1:2000 dilution, respectively;immunolocalization was achieved by the use of alkalinephosphatase-conjugated goat anti-rabbit IgG (Promega) ac-cording to the manufacturer's recommendations.cDNA Library. Five micrograms of liver poly(A)+ RNA

prepared from a hibernating Richardson's ground squirrelwas used for library construction in AZAPII (Uni-ZAP kit,Stratagene). The library contained 1.6 x 106 plaque-formingunits before amplification; 98% of the phage were recombi-nants.RNA Blots and Hybridization Probes. Total RNA isolated

from the livers of active and hibernating animals (13), wasapplied to nitrocellulose by Northern blotting (14) or with aslot blot apparatus (BioRad). Blots were hybridized to 32p-labeled cDNA (prepared by the random-primer method witha kit from Boehringer Mannheim) to either ground squirrela2-macroglobulin, in 50%6 formamide, or mouse albumin (15),in 40%o formamide (14). For slot blots, all samples wereapplied in triplicate; radioactivity remaining on the blot afterwashing [3 x 5 min at room temperature in 2x standard salinecitrate (SSC)/0.1% SDS, followed by 2 x 15 min at 420C in0.8x SSC/0.1% SDS) was determined directly (Bioscanimaging scanner system 200-IBM). The a2-macroglobulinradioactivity for each sample was averaged and then normal-ized to the average albumin radioactivity.

RESULTS AND DISCUSSIONPlasma samples were taken from a number ofhibernating andactive individuals of Richardson's ground squirrel for deter-mination of albumin concentrations. Results from dye bind-

ing assays for albumin and total protein determinations arepresented in Table 1. These results demonstrate an absoluteincrease in the concentration ofalbumin during hibernation inRichardson's ground squirrel, as has been reported previ-ously for hamsters (6) and 13-lined ground squirrels (7). Whenthe concentration of albumin is compared with the totalprotein concentration, however, there is no relative increasein albumin during hibernation (Table 1). This suggests thatthe observed increase in albumin concentration during hi-bernation is not an actively regulated process but rather apassive response to some factor that affects all of the plasmaproteins simultaneously. A likely candidate for such a non-specific factor is the dehydration that naturally accompanieshibernation.

Since the dye binding assays used to obtain the results ofTable 1 were developed and standardized for human samples,the plasma proteins were also examined by SDS/PAGE.When a constant amount of protein was applied to each lane,the amount of albumin did not vary between the active andhibernating animals (Fig. 2A). This confirms the resultsobtained with the dye binding assays. Interestingly, on thesame gels, an unknown protein with a molecular size of '170kDa was observed at consistently elevated amounts in sam-ples prepared from hibernating animals relative to samplesprepared from active animals. This protein was isolated andused to immunize rabbits. The resulting antiserum, but notthe preimmune serum, detected the 170-kDa plasma proteinon Western blots (Fig. 2B).The antiserum to the 170-kDa protein was also used to

screen a cDNA library that was prepared in AZAPII using

Table 1. Albumin and protein concentrations in Richardson'sground squirrel plasma

No. of Albumin, Protein, Albumin as %State animals mg/ml mg/ml of protein

Active 9 23 6 51 11 45 ± 4Fall-active 6 28 3 64± 5 43 ± 3Hibernating 9 30 3* 66 6** 46 + 5***

Percent ofalbumin in total protein was calculated for each sample,and then the average of those values was determined. P values foractive/hibernating comparison determined by Student's t test: *,0.01 <P < 0.05; **, 0.001 <P < 0.01; ***, 0.50 <P < 0.70. All ofthe animals from groups A and B are represented here (see Materialsand Methods).

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A1 2 3

_P.iM__-

4 5 6 7 8 9 10 11is.. - :..:r,-:w

B _- -_ _ Mp _

C

Albumin

1 2 3 4 1 2 3 4 -ori

ow--" -28S-18S

FIG. 2. Albumin protein and mRNA levels remain cowhile the 170-kDa protein (a2-macroglobulin) and mRNAincrease during hibernation. (A) Coomassie blue-staine'polyacrylamide gel (7.5% acrylamide) with 7.5 1Lg of proteirin each lane. Only the region of the gel around 68 kDa is shcmajor band is albumin. Based upon its apparent molecularrelative abundance in the plasma, the lighter band(s) jusalbumin is transferrin, which shows a polymorphism in thilation of Richardson's ground squirrels. Lanes 1-6, plasnRichardson's ground squirrels (group A animals); lanes 7-10,from Columbian ground squirrels (group C animals); lane 11,proteins (Bethesda Research Laboratories; the sizes ofmarker bands are given in kDa). Lanes 1-3, 7, and 8 are froranimals; lanes 4-6, 9, and 10 are from hibernating animWestern blot of the top of the same gel as in A, showing theabove transferrin after transfer to nitrocellulose (LKB Novabimmunostaining. (C) Northern blot of Columbian groundliver mRNA. Lanes contained 10 ,Ag of total RNA isolated flivers of the same Columbian ground squirrels as used in ALanes 1 and 2, active animals; lanes 3 and 4, animalshibernation. Duplicate blots were hybridized to either albia2-macroglobulin (a2M) cDNA as indicated. Positions of28SrRNA, visualized by UV shadowing before transfer to nitroceare marked. ori, Origin.

mRNA isolated from the liver of a hibernating Richarground squirrel. Six clones that reacted with the anwere isolated, with inserts ranging from 2.6 to 4.4 kil(kb) in length. Based on their restriction maps and atcross-hybridize on Southern blots, all six cDNA insederived from a single mRNA species. The DNA seifrom both ends of each insert nearest the vector wasmined (available from the authors upon request), leassequence from five separate regions (some of the seqoverlapped) encompassing 1277 nucleotides. Of thes(nucleotides lie within the protein-coding region andspond to a total of 350 amino acids. The derived amiisequences and the FASTA program (16) were used tothe Swiss-Prot data base (September 1990). All of the Isquirrel sequences showed the greatest sequence i((70-80%) to previously published amino acid sequela2-macroglobulin from human (17) and rat (18), si

suggesting that the plasma protein that increaseshibernation is a2-macroglobulin. The amino acid sequea CNBr cleavage fragment generated from FPLC-p170-kDa protein matched the derived amino acid se(

from a region of the cDNA sequence that lies just down-stream of a methionine codon, thereby confirming that theclones isolated with the antiserum correspond to the proteinthat was initially observed by SDS/PAGE (data not shown).

-71 a2-Macroglobulin is a broad-spectrum protease inhibitorthat functions by "caging" its protease substrate, then caus-ing its elimination from the circulation. The caged proteasesretain their activity toward small-molecule substrates, form-ing the basis of an in vitro assay (10), which we used toquantitate the levels of a2-macroglobulin in a number of

-106 plasma samples taken from ground squirrels (Table 2). Thesame laboratory-housed active and hibernating Richardson's(group A, Table 2) and Columbian (group C) ground squirrelsas used for the data in Table 1 and Fig. 2 A and B wereassayed. As described in Materials andMethods, plasma wasprepared from active and hibernating animals in groups A andC on the same day; thus they are state-dependent andseason-independent. In addition, the assay was performed onplasma prepared from a number of wild-caught Richardson'sground squirrels in early summer or late fall (Group B,season-dependent). The specific activity of a2-macroglobulinis highest in the hibernating animals and lowest in the activeanimals (Table 2), with the interesting exception of those thatwere sampled in the fall (see below). During hibernation, theRichardson's and Columbian ground squirrels have a 2.5-foldand a 3.7-fold elevation in a2-macroglobulin activity, respec-

onstant, tively.k levels The mean activity of a2-macroglobulin in the six "late fall"d SDS/ animals is intermediate with respect to the active and thei loaded hibernating values (Table 2). These six animals also show theiwn; the widest individual variation that we have observed in the leveliass and of a2-macroglobulin activity (note the standard deviation

Itabove values in Table 2), such that some of the animals are close to

s popu-the active value and some are close to the hibernating value.

na fromThis is best explained by the fact that, in the wild, individual

marker animals will enter hibernation at different times over thevisible course of a month (19). Since these six animals were trapped

m active and sampled in mid-October, they were expected to be atals. (B) different stages in their preparation for hibernation. Thee region variable levels of a2-macroglobulin activity in the fall animalslot) and also provide evidence that the increase in a2-macroglobulinsquirrel during hibernation is not merely a consequence of becoming

oand B. hypothermic, because these animals were euthermic at thein deep time of sampling. Rather, the observed increase in a2-imin or macroglobulin appears to be the result of an actively con-and 18S trolled process that begins when the animal prepares tollulose, hibernate. In addition, the increase in a2-macroglobulin does

not appear to be a seasonal fluctuation that is independent ofhibernation, because the increase in a2-macroglobulin activ-

rdson's ity during hibernation was observed when plasma was pre-

itibody pared from animals that were active or hibernating on theobases same day in the laboratory (groups A and C). In the Rich-

,ility to ardson's ground squirrels, the numbers obtained from plasmaXrt5 are collected on the same day (three active, three hibernating,quencedeter-

ding touencese, 1050corre-no acidsearchgrounddentitynces oftronglyduringence ofurifiedquence

Table 2. Comparison of a2-macroglobulin activity in active andhibernating ground squirrels

Inhibitor units/No. of tug of protein, Normalized

Group animals State value x 105 to activeRichardson's 3 Active 6.16 ± 1.06 1.00group A 3 Hibernating 15.60 ± 4.58 2.53*

Richardson's 6 Active, summer 6.67 ± 1.59 1.00group B 6 Active, late fall 12.30 ± 6.71 1.84

6 Hibernating 16.20 ± 6.28 2.43**Columbian 2 Active 13.40 ± 4.77 1.00group C 2 Hibernating 49.60 ± 4.44 3.70*P values determined by Student's t test: *, 0.01 < P < 0.05; **, P

= 0.001.

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Group A animals) are quantitatively similar to the numbersobtained for the summer-active/winter-hibernating (six each)animals sampled seasonally, on various dates (group B; Table2).These results are consistent with the hypothesis that a2-

macroglobulin, in contrast to albumin, is increased by aspecific mechanism as the animal prepares to hibernate, andis maintained at elevated levels throughout hibernation. Theamount of circulating a2-macroglobulin can be increasedeither by an increase in the rate of synthesis of the protein orby a decrease in its rate of degradation. A common mecha-nism used to increase the rate of synthesis of a protein is toincrease the amount of its corresponding mRNA, often viaincreased transcription. To address the mechanism of controlof a2-macroglobulin expression during hibernation, thecloned ground squirrel a2-macroglobulin cDNA was used asa hybridization probe on Northern or slot blots containingtotal RNA prepared from active and hibernating groundsquirrel livers.An mRNA of about 4.8 kb in liver RNA from both active

and hibernating Columbian ground squirrels was found tohybridize to the a2-macroglobulin cDNA probe. The length ofthis mRNA is sufficient to encode a 170-kDa polypeptide andagrees well with the known length of the a2-macroglobulinmRNA from rat (18). An increase in the amount of a2-macroglobulin mRNA in the liver from hibernating animalsrelative to active ones was readily observed, in contrast toalbumin mRNA which remained constant between the twostates (Fig. 2C). Slot blot analysis was used to quantitate therelative increase of a2-macroglobulin mRNA during hiberna-tion in a number of liver RNA samples prepared fromColumbian and Richardson's ground squirrels (Table 3). Theincrease in armacroglobulin mRNA during hibernation ismore than sufficient to account for the increase in a2-macroglobulin protein and activity in both species. Thisresult is consistent with a specific, regulated increase in therate of a2-macroglobulin mRNA synthesis or stability duringpreparation for, and maintenance of, the hibernating state.An independent example of a specific, regulated increase

in a2-macroglobulin mRNA and protein levels during anenvironmental challenge to the animal is found in the acute-phase response of the rat. This response occurs duringinflammation, which may be caused by a variety of homeo-static challenges to an organism, including infection, trauma,immunological disorders, and neoplastic growth (20). Theacute-phase response involves both increases and decreasesin the expression of a number of liver-specific proteins. Inrats, the increase in circulating a2-macroglobulin following aninflammatory stimulation parallels the 200-fold increase inthe level of liver a2-macroglobulin mRNA. Between 2.3- and4-fold of this increase is due to an increase in the rate oftranscription (18, 21). Interestingly, a2-macroglobulin is notan acute-phase protein in all species (in humans, for in-stance). It is not known whether a2-macroglobulin is anacute-phase protein in ground squirrels, nor is it knownwhether armacroglobulin plays a general or a specific roleduring hibernation.

Table 3. Comparison of a2-macroglobulin (a2M) mRNA levels inactive and hibernating ground squirrels

No. of a2M/albumin NormalizeSpecies animals State mRNA to active

Richardson's 3 Active 0.55 ± 0.24 1.003 Hibernating 3.87 ± 1.69 7.03*

Columbian 2 Active 0.75 ± 0.22 1.002 Hibernating 3.22 ± 0.48 4.28**

P values determined by Student's t test: *, P = 0.01; **, 0.01 < P< 0.05.

Environmental Signals; Endogenous Circannual Rhythm

BRAIN 4> Modify Gene Expression

TISSUES, including LIVER =2 Modify Gene Expression

(PHYSIOLOGICAL CHARACTERISTICS OF HIBERNATION)

FIG. 3. Model depicting the central role of differential geneexpression during hibernation. Single arrows indicate direct effects,whereas multiple arrows indicate an effect that requires multipleintermediate events (see text).

Animals in hibernation generally exhibit surprisingly littleevidence of protein degradation, as if there were a wide-spread inhibition of proteolysis (1). This is thought to be animportant adaptation for hibernation both because the abilityof the animals to eliminate urea is impaired and because theanimals need to be ready to function for reproduction imme-diately upon emergence in the spring (1). Since a2-macroglobulin is capable of inhibiting a broad spectrum ofproteolytic enzymes, it may be increased during hibernationto reduce proteolysis in a general way. Alternatively, a2-macroglobulin may play a specific role in the inhibition ofblood clotting; a2-macroglobulin is known to be the mainphysiological inhibitor ofactivated factor X, a key enzyme inboth the intrinsic and extrinsic clotting pathways (22). It maybe important to reduce clotting during hibernation because ofthe low heart rate.The identification of a protein, a2-macroglobulin, that

increases specifically for hibernation is consistent with ourhypothesis that the adaptation of hibernation evolved at amolecular level by changes in patterns of gene expression.Although the liver is likely to be only one of many tissueswhere differential gene expression occurs in a hibernatingmammal, it is apt to play a major role in survival of thehomeostatic challenge provided by hibernation. A possiblescheme, depicting a more global role for differential geneexpression in the specification of the hibernating phenotype,is shown in Fig. 3. Internal signals and/or signals from theenvironment converge in the brain, where they lead to achange in gene expression. These gene products in turn,acting either directly or indirectly, lead to a repatterning ofgene expression in a number of tissues throughout the body,as demonstrated here for the liver. All of these changesultimately add up to the constellation of physiological traitsthat characterize hibernation.Mammalian hibernation provides an ideal model system to

study the role of differential gene expression in adaptiveevolution. Although several studies have provided evidencefor the regulatory hypothesis of molecular evolution (23-26),hibernation is, to our knowledge, the first example of abehavioral phenotype that has been examined in this context.Further studies aimed at elucidation of the details of themolecular pathways outlined in Fig. 3 will clarify how com-plex adaptive behaviors arise during evolution. In addition, itmay be possible to work backwards from the hibernation-induced differential expression of liver genes, such as that ofa2-macroglobulin, demonstrated here, to the putative mastercontroller-i.e., the elusive (27, 28) trigger molecule (29)--ofhibernation.

We thank D. Belke and J. Westly for tissues, J. Crosby and D.Branciforte for preparation ofantiserum, G. Stetler and P. Cozart forhelp with purification of Richardson's ground squirrel a2-

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macroglobulin, J. Lippincott for protein microsequence determina-tion, and A. Franzusoff, K. Howell, M. Lohka, S. Nordeen, K.Thomas, and E. Zimmer for helpful discussion. This work wassupported by Biomedical Research Support Grant 05357, Hepato-biliary Research Center P30-DK34914, and Army Research OfficeGrant DAAL03-92-G-0019 to S.L.M. and by Natural Sciences andEngineering Research Council Grant (Canada) Grant A6455 toL.C.H.W.

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