ORIGINAL PAPER

Mauricio Berriel Diaz Æ Martin Lange

Gerhard Heldmaier Æ Martin Klingenspor

Depression of transcription and translation during daily torporin the Djungarian hamster (Phodopus sungorus)

Accepted: 27 May 2004 / Published online: 30 June 2004� Springer-Verlag 2004

Abstract During daily torpor, Djungarian hamstersreduce their metabolic rate by more than 70% belowtheir resting metabolic rate for several hours per day. Weinvestigated whether this depression of metabolism isassociated with a reduction in transcription and trans-lation. Liver tissue was sampled in defined metabolicstates: during normometabolism, in the torpid state andafter arousal from torpor. Nuclei were isolated fromliver tissue and subjected to nuclear run-on assays at anassay temperature of 25 �C. We observed a �40%decrease in transcriptional initiation in liver nuclei ofhamsters which had attained minimal metabolic rateduring torpor as compared to nuclei from normometa-bolic hamsters. During arousal from torpor, thetranscriptional run-on activity recovered to the normo-metabolic level. Polysome profile analysis of liver tissuewas used to determine the proportion of actively trans-lating polysomes. Profiles of liver samples from torpidanimals show a disaggregation of polysomes comparedto profiles from normometabolic hamsters, which indi-cates that, in addition to transcription, protein synthesisdecreases during torpor. These results indicate thatduring torpor a specific inhibition of the energeticallycostly processes of RNA and protein synthesis contrib-utes to the overall metabolic depression.

Keywords Daily torpor Æ Transcription ÆNuclear run-on assay Æ Translation Æ Polysome profiles

Abbreviations L:D: light:dark phase Æ RMR: restingmetabolic rate Æ RQ: respiratory quotient Æ VCO2:carbon-dioxide production Æ VO2: oxygen consumption

Introduction

Daily torpor and hibernation are characterized by adecrease in heart rate, metabolic rate and body tem-perature, resulting in a reduction in daily energyexpenditure, and providing a strategy for coping withlimited food availability and cold. Hibernation boutscan be maintained for several days or weeks, whereasdaily torpor bouts are restricted to the daily period ofrest. Djungarian hamsters (Phodopus sungorus) fed adlibitum exhibit spontaneous bouts of daily torpor inwinter, when acclimated to short photoperiods (Held-maier and Steinlechner 1981; Heldmaier et al. 1989a).This torpor behavior is part of seasonal acclimationtriggered by short photoperiods, which also involvesother morphological and physiological adaptations,such as reduced body weight, increased capacity fornonshivering thermogenesis, improved fur insulation,white fur coloration and reproductive quiescence(Heldmaier et al. 1989b; Heldmaier and Klingenspor2002).

In daily torpor, metabolic rate is reduced by morethan 70% of RMR (Geiser 1988; Heldmaier and Ruf1992). Daily torpor provides a good model for studyingmolecular processes associated with hypometabolism. Instrictly photoperiodic species like the Djungarian ham-ster, spontaneous daily torpor occurs due to shortphotoperiod exposure at predictable times of day, anddoes not require other stimulations like cold treatmentand food restriction. Also the degree of hypothermia isless pronounced than in hibernators, which reduces theeffect of low temperature on biochemical processes.

At an ambient temperature (Ta) of 15 �C, bodytemperature (Tb) is reduced to a minimum of about20 �C (Heldmaier et al. 1999). Simultaneous recordingsof metabolic rate and Tb during the phase of entranceinto torpor clearly demonstrated that a rapid decrease inmetabolic rate precedes the development of hypother-mia. This time course as well as changes in metabolicenzyme activities suggest that the entrance into the

Communicated by L.C.-H. Wang

M. Berriel Diaz (&) Æ M. Lange Æ G. HeldmaierMartin KlingensporFachbereich Biologie, Tierphysiologie,Philipps Universitat Marburg, Karl von Frisch Str. 8,35043 Marburg, GermanyE-mail: berrield@staff.uni-marburg.deTel.: +49-6421-2825372Fax: +49-6421-2828937

J Comp Physiol B (2004) 174: 495–502DOI 10.1007/s00360-004-0436-2

hypometabolic state involves active metabolic depres-sion (Snyder and Nestler 1990; Storey and Storey 1990;Heldmaier and Ruf 1992; Malan 1993; Storey 1997;Heldmaier et al. 1999).

Several studies on hibernators have addressed theidea that a reversible depression of the energeticallyexpensive processes of transcription and translation maysignificantly contribute to the observed decline in met-abolic rate during deep hibernation (Whitten and Klain1968; Gulevsky et al. 1992; Zhegunov et al. 1992;Frerichs et al. 1998; Knight et al. 2000; van Breukelenand Martin 2001, 2002). Recent studies on hibernatinggolden-mantled ground squirrels by van Breukelen andMartin show a reduction in the level of transcriptionalinitiation in early hibernation bouts by 28% comparedto the euthermic rate and almost 50% of the arousal rate(van Breukelen and Martin 2002), and a severe reduc-tion in translational initiation at body temperaturesbelow 18 �C (van Breukelen and Martin 2001). Weinvestigated whether such changes in transcription andtranslation occur in Djungarian hamsters entering dailytorpor under conditions where minimal metabolic rate isfirst attained and body temperature is above 20 �C.

Material and methods

Animals

Djungarian hamsters were bred and raised at our insti-tute in a long photoperiod (16:8 h L:D) at 23 �C ambi-ent temperature (Ta) and had free access to food andwater. When hamsters reached the age of 6 weeks, theywere acclimatized to a short photoperiod (8:16 h L:D) at15 �C with ad libitum food and water. After 6–8 weeksof exposure to short photoperiod, hamsters started todisplay daily torpor. An ambient temperature of 15 �Cwas chosen since acclimation occurs fastest and ham-sters show highest incidence of torpor under these con-ditions.

Metabolic rate and tissue sampling

O2 consumption and CO2 production were measuredcontinuously for several days using an open-flow indi-rect calorimetry system (Heldmaier and Ruf 1992) inorder to monitor the frequency, timing and depth oftorpor in each individual hamster. Tissues were sampledin defined metabolic states. Hamsters were sacrificedeither in the normometabolic state on a day they did notenter torpor (normometabolic), when minimum meta-bolic rate during torpor was first attained (torpid), orafter arousal from torpor, when metabolic rate hadreturned to normometabolic values (aroused). For nor-mometabolic samples, the time of sacrifice correspondedto the time when minimal metabolic rate during torporhad been attained on a previous day. Hamsters werekilled by cervical dislocation. Tissues were rapidly

dissected, transferred to liquid nitrogen and stored at�80 �C until analysis. All subsequent procedures wereperformed under conditions to avoid RNAse contami-nation.

Resting metabolic rate (RMR) was determined as themean value (±SEM) of the eight lowest records of O2

consumption in the normometabolic state during thediurnal resting period before sacrifice. For torpid ham-sters, the time before the torpor episode was used todetermine RMR. The onset of daily torpor is preceded bya characteristic peak in metabolic rate, followed by adecrease in metabolic rate below the level of RMR. Thispeak was defined as the beginning of entrance into torpor.

Isolation of nuclei from liver tissues

Liver tissue was pulverized under liquid nitrogen andhomogenized in 5 ml of 10 mM Hepes, pH 7.6, 25 mMKCl, 0.6 mM spermine, 2 mM spermidine, 1 mMEDTA, and 40% glycerol with a Dounce homogenizer(6 strokes with the loose type, 101.0 lm clearance; 2strokes with the tight type, 50.8 lm clearance). Thehomogenate was filtered through nylon gauze (60 lm)and pelleted at 900 g at 4 �C for 5 min using a swingingrotor in a Beckman J2-21 centrifuge. The pellet wasresuspended in 2.5 ml of 10 mM Hepes, pH 7.6, 25 mMKCl, 0.3 mM spermine, 1 mM spermidine, 1 mMEDTA, 10% glycerol, and mixed with 2.5 ml of 50%iodixanol solution (OptiPrep, Nycomed Pharma, Nor-way). This solution was laid on top of a 0–35% iodix-anol gradient and centrifuged at 10,000 g at 4 �C for20 min. Nuclei were separated from other cell compo-nents and harvested from a white layer in the gradient.Nuclei were subsequently washed in 10 ml of ice-cold10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 10 mM NaCland centrifuged at 900 g, 4 �C for 5 min using aswinging rotor in a Beckman J2-21 centrifuge. The pelletwas resuspended in 500 ll of 50 mM Tris-HCl, pH 8.3,5 mM MgCl2, 0.1 mM EDTA, 40% glycerol, and snap-frozen in liquid nitrogen. The nuclei were stored at�80 �C until use.

Determination of nuclear densities and quality control

The number of nuclei was determined by fluorometricquantification of DNA concentrations. A standardcurve was generated measuring the fluorometric signalof defined concentrations of calf thymus DNA in Hoe-chst dye 33258 (100 ng/ml in storage buffer). Subse-quently, the fluorometric signal of 1 ll of nuclearpreparation in Hoechst dye was determined and thenumber of nuclei calculated assuming that the DNAconcentration of a single nucleus is 6 pg (Capparelliet al. 1997). Typical preparations resulted in a yield of2·107 nuclei/g liver.

The results of the fluorometric quantification wereconfirmed by a second method in which a defined

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aliquot of nuclei was incubated in Hoechst dye 33258(100 ng/ml). Nuclei were subsequently counted using acounting chamber (Thoma) in a fluorescence microscope(excitation 350 nm, emission 450 nm). Both methodsresulted in similar nuclear densities (data not shown),thus the faster method of fluorometric quantificationwas subsequently used.

The quality of the isolated nuclei was observed usinga confocal laser scanning microscope (Leica, TCS SP2).Briefly, an aliquot of nuclei was incubated with BOPRO-3 (Molecular Probes) for 10 min. The integrity of thenuclei was checked by adding a small volume of nuclei toa counting chamber and by subsequent observationunder the microscope (excitation 575 nm, emission599 nm).

Nuclear run-on assay

Assays were performed according to van Breukelen andMartin (2002) with slight modifications. All nuclear run-on assays were performed at 25 �C for 15 min in a totalassay volume of 170 ll containing 47.8 mM Tris-male-ate, pH 7.6, 71.6 mM KCl, 19.1 mM (NH4)2SO4,2.3 mM MgAc, 14.3 mM dithiothreitol, 1.9 mMMnCl2, 10 U RNAsin (Promega), 44 ll storage buffer(containing 50 mM Tris-HCl, pH 8.3, 5 mM MgCl2,0.1 mM EDTA, 40% glycerol), 240 lM ATP, 480 lMCTP, 480 lM GTP, 48 lM UTP, 5 ll of 111 TBq/mM32P-UTP (185 MBq) and 40 ll of a nuclear suspensioncontaining 2.75·107–5.0·108 nuclei/ml. In each experi-ment, nuclei from normometabolic hamsters were incu-bated in a heat block, simultaneously with equalnumbers of nuclei from either torpid or aroused ham-sters. Aliquots of 15 ll were taken from the mixtures atdefined time points (1, 5, 10 and 15 min) in order tomonitor the in vitro incorporation rate over time. Forthe comparison of the in vitro transcription rates ofdifferent samples, only the 32P-UTP incorporation after10 min of incubation (maximal incorporation) was used.The aliquots were streaked out onto glass-fiber filters(Schleicher und Schuell, GF6). Filters were immediatelywashed in 10% trichloric acid, 1% sodium acetate for60 min; twice in ice-cold 5% trichloric acid, 0.5%sodium-acetate for 10 min; and three times in 95%EtOH for 10 min. Filters were subsequently dried andthe remaining radioactivity was measured by scintilla-tion counting (Beckman LS 3801).

Polysome profiles

Sucrose-gradient preparation and fractionation forpolysome profile analysis were performed as describedpreviously (van Breukelen and Martin 2001) with someslight modifications. Approximately 0.4 g of frozen andpulverized liver tissue (including some samples that werealso used for run-on assays) were homogenized in 5 volof ice-cold 25 mM Tris-HCl, pH 7.6, 25 mM NaCl,

10 mM MgCl2, 250 mM sucrose, 1 mg/ml heparin, and0.1 mg/ml cycloheximide with six passes of a Dounce B(clearance 50–70 lm) pestle and three passes with aDounce A (clearance 10–30 lm) pestle. The homogenatewas centrifuged for 15 min at 16,000 g at 4 �C. Thesupernatant was adjusted to 0.5% each of sodium de-oxycholat and Triton-X, and carefully layered on top ofa 20–60% sucrose gradient containing 10 mM Tris-HCl,pH 7.6, 300 mM NaCl, 10 mM MgCl2, 0.1 mg/ml hep-arin, and 0.1 mg/ml cycloheximide. The samples werecentrifuged for 3 h at 235,000·g at 4 �C in a swingingbucket rotor (SW-50, Beckman). The gradients wererecovered in 400-ll fractions by manual collection andsnap-frozen in liquid nitrogen.

Northern blot analysis

Total RNA was isolated from the individual gradientfractions using Trizol LS (GIBCO/Invitrogen). RNAwas separated on a 0.8% agarose–formaldehyde gel. Theethidium bromide-stained RNA was visualized on a UVscreen and photographed for densitometric quantifica-tion before being transferred to a nylon membrane(Hybond N+, Amersham) by capillary force (downb-lotting). The image of ethidium bromide-stained RNAwas generated without areas of optical saturation toassure a linear relationship between signal intensity andamount of RNA. For Northern blot analysis, a frag-ment of the actin cDNA that previously had been clonedin our laboratory from hamster tissue was radiolabeledusing the Redi-prime kit (Amersham Pharmacia) andhybridized to the blots. Actin signals on Northern blotswere quantified densitometrically by phosphorimageanalysis (ImageQuant, Molecular Dynamics).

Results

Metabolic rate of Djungarian hamsters was recordedcontinuously for several days. Hamsters performed dailytorpor spontaneously about every second day under theexperimental conditions chosen for this study. Dailytorpor was characterized by a drop in metabolic ratebelow the level of resting metabolic rate. All hamstersshowed a similar time course of entry into and arousalfrom torpor. Liver tissue was sampled either in thenormometabolic state at the same time hamsters hadperformed torpor on previous days, or in the torpid statewhen hamsters first reached minimum metabolic rate, orafter arousing from torpor (Fig. 1). O2 consumption,CO2 production and respiratory quotient (RQ) ofhamsters at the time of sacrifice are listed in Table 1.Metabolic rate of torpid hamsters was lowered by morethan 70% below their RMR. Hamsters sacrificed in thetorpid state reached minimum metabolic rate 3.2±0.3 h(n=6) after the initial metabolic peak initiating thetorpor bout.

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RQ was lowered from �1 before and during entranceinto torpor to 0.75±0.02 in aroused hamsters (Table 1).This is in accordance with previous measurements(Heldmaier et al. 1999), where a high respiratory quo-tient (RQ�1) at the beginning of torpor steadily de-creased during hypometabolism, reached minimal valuesjust before arousal and returned to pretorpor levelwithin 2 h after arousal.

Nuclear run-on assays

The incorporation of radiolabeled ribonucleotides intonascent RNA chains in vitro represents the degree oftranscriptional initiation in vivo. As transcriptional ini-tiation in vitro is reduced to very few genes (Weber et al.1977), it is possible to take a snapshot of the transcrip-tional activity in vivo at the time of tissue removal. Livernuclei obtained from normometabolic, torpid andaroused hamsters were assayed in pairs at an assaytemperature of 25 �C, comparing hepatic nuclei fromnormometabolic hamsters with nuclei from torpid oraroused hamsters. Aliquot samples out of every assaywere taken after 1, 5, 10 and 15 min of incubation. 32P-UTP incorporation was observed over a period of10 min. No further incorporation occurred after 15 minof incubation. This may be due to run-off of pre-initi-ated transcripts and/or the high abundance of endoge-nous RNAses in the liver. The time scale is inaccordance with earlier observations of nuclear run-onassays from liver nuclei (Adams and Goodman 1976;van Breukelen and Martin 2002). Only the maximumincorporation of 32P-UTP after 10 min was used for thecomparison of samples. The incorporation data after 1,5 and 15 min are not shown. The amount of incorpo-rated radioactivity measured using scintillation countingwas normalized to 107 nuclei. To test whether allradioactivity bound to filters was incorporated intopolyribonucleotides, RNA polymerization was eitherinhibited by a-amanitin or by replacement of rNTP withdNTP in the reaction. Comparing nuclei from identicalpreparations revealed that 0.05% (n=4) of total radio-activity in the assay bound unspecifically to the filter andwas therefore not due to RNA synthesis. Therefore, ineach assay, this proportion of total counts was sub-tracted from counts measured on filters as background.

Nuclei from torpid hamsters showed a 39.8%reduction in incorporation of radiolabeled nucleotidesafter 10 min of incubation compared to nuclei from thecorresponding normometabolic controls (Fig. 2; n=3;t-test P<0.001). Nuclei from aroused hamsters did notdiffer in incorporation from their normometabolic con-trols (Fig. 2; n=4).

Polysome profiles

Actively translating ribosomes are typically associatedwith transcripts forming polysomal complexes. By cen-

Fig. 1a–c O2 consumption (metabolic rate) of normometabolic,torpid and aroused hamsters. Each panel contains two metabolicrate measurements for the same hamster. Solid lines Metabolic rateon the day of sacrifice, dashed lines measurements for 24 h whenhamsters performed torpor, asterisks O2 consumption at the timeof sacrifice. Tissue sampling in a the normometabolic state, b thetorpid state, c the aroused state. The black bar indicates the darkphase from 4 p.m. to 8 a.m.

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trifugation of a tissue homogenate within a sucrosegradient and subsequent fractionation of the gradient, itis possible to quantify the distribution of ribosomespresent as polysomes or monosomes. Material (mRNAand rRNA) that is found at the high-density lower endof the gradient represents actively translating polysomes.A shift in the position of both mRNA and rRNA in thegradient shows a disaggregation of polysomes indicatinga global depression of protein synthesis in the tissueexamined.

Denaturing agarose gel electrophoresis was per-formed on the RNA isolated from the fractionatedgradients and revealed a reduction in 18S and 28S rRNAas well as mRNA in the polysome-containing end of thesucrose gradient and a shift of material to the middle ofthe gradient in samples from liver tissue of torpidhamsters as compared to normometabolic hamsters(Fig. 3a). This effect was quantified by densitometrywithin the linear range of ethidium bromide-stainedRNA from four normometabolic and four torpid ham-sters (Fig. 3c). On Northern blots, we investigatedwhether, in the torpid state, an altered distributionwithin the gradient can also be observed for the repre-sentative transcript actin (Fig. 3b). In samples fromnormometabolic hamsters, densitometric quantificationrevealed the exclusive presence of actin mRNA inpolysomal fractions, whereas in samples from torpidhamsters a significant portion of the total actin signal

intensity appears in the middle of the gradient. Still alarge portion of total actin mRNA is found in the po-lysomal region of the gradient in torpid hamsters(Fig. 3d).

Discussion

Transcriptional run-on assays were employed to inves-tigate the status of transcriptional initiation as a func-tion of the torpid state. The determination of the in vitrotranscription rate in isolated nuclei reflects a snapshot ofglobal transcriptional activity in a given physiologicalstate. In isolated nuclei, no de novo initiation has beenreported except for 5S rRNA genes which can transcribeup to three copies in 40 min (Yamamoto and Seifart1977). The transcription rate of a certain gene is acombination of initiation and elongation. The rate ofpolymerization of all three eukaryotic RNA polymerasesis similar, so differences in the amount of gene products(transcripts) at a constant temperature are strictly due todifferences in the status of transcriptional initiation atthe time of tissue removal. All experiments were con-ducted at 25 � C. At this temperature the RNApolymerases were sufficiently active but otherwiseRNAse activity remained low. Results clearly demon-strate that hypometabolism during daily torpor is asso-ciated with a reduction in transcriptional initiation,similar to the extent of reversible depression of tran-scription shown for hibernation (van Breukelen andMartin 2002).

Parallel measurements of metabolic rate and bodytemperature of Djungarian hamsters during daily torporat Ta=15 �C demonstrate that hamsters attaining min-imum metabolic rate during entrance into torpor, as inthe present study, had a body temperature of23.2±0.4 �C (n=8) (Heldmaier et al. 1999). Hypother-mia will further slow down the biochemical machineryof gene expression because of the temperature sensitivityof transcriptional elongation. Temperature-dependencyexperiments have shown that a reduction in assay tem-perature also leads to decreased transcription rates invitro based on the expected Q10 of 2–3 (van Breukelenand Martin 2002). However, in hibernators a seasonaladaptation of the transcriptional machinery to low bodytemperatures does not occur, as nuclei from summer-and winter-acclimated, non-hibernating squirrels as well

Fig. 2 Left bars State-specific run-on activity for normometabolic(N; n=3) and torpid (T; n=3) hamsters after 10 min of incubation.Asterisk Means are significantly different (t-test for paired samplesP<0.001). Right bars State-specific run-on activity for normomet-abolic (N; n=4) and aroused (A; n=4) hamsters after 10 min ofincubation. Values represent means±SEM of 32P-UTP incorpora-tion in lmoles normalized to 10

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nuclei

Table 1 Metabolic characterization of normometabolic and torpidand aroused states. Oxygen consumption (VO2), carbon dioxideproduction (VCO2) and respiratory (RQ) of normometabolic (N),torpid (T), and aroused (A) hamsters correspond to the time ofsacrifice for tissue sampling. Resting metabolic rate (RMR) was

determined as the mean value of the eight lowest measurements ofO2 consumption of individual hamsters in the normometabolicstate. Time in torpor was defined as the time from the initial met-abolic peak to the time of sacrifice (T) or to the time metabolic raterose above RMR (A). Values shown are means±SEM

State Body weight (g) VO2 (ml h–1) VCO2 (ml h–1) RQ Time in torpor (h) RMR at 15 �C (ml O2 h–1)

N (n=9) 25.2±0.9 76.4±4.9 77.7±4.3 1.03±0.03 - 55.0±1.57T (n=6) 25.0±1.4 16.7±2.0 15.2±1.5 0.92±0.05 3.2±0.4 53.4±2.9A (n=4) 25.4±1.6 97.4±18.6 73.7±14.8 0.75±0.02 5.6±0.4 58.0±3.2

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as rats showed similar activities at different assay tem-peratures (van Breukelen and Martin 2002).

We conclude that combined effects of down-regulatedtranscriptional initiation and temperature-dependentreduction in elongation rates lead to transcriptionaldepression during torpor. This is in accordance withresults from in vivo experiments. The incorporation ofradiolabeled nucleotides into acid-precipitable materialin brains of hibernating ground squirrels has been shownto be reduced by 90% (Frerichs et al. 1998).

Unlike nuclei from squirrels aroused from hiberna-tion bouts (van Breukelen and Martin 2002), the tran-scription rates of nuclei from hamsters aroused fromdaily torpor showed no difference compared to thenormometabolic level. While a hibernation bout can lastfor several weeks, daily torpor is reduced to a period ofseveral hours. Thus, it is questionable whether there is aneed for increased mRNA production to replenish RNApools after a daily torpor bout. Furthermore, the de-creased utilization of transcripts for translation, andmechanisms like degradation-protective binding ofpolyA-binding proteins (PABP) to mRNA (Knight et al.2000) may contribute to the stabilization of the mRNApool during torpor.

Transcription contributes about 5% to oxygen con-sumption rate (Rolfe and Brown 1997). Regarding thesevere metabolic depression of about 70% of RMRassociated with daily torpor (Heldmaier and Ruf 1992),it is likely that other processes like translation must alsobe reduced. Protein synthesis requires 25–30% of ATP-fixed energy (Rolfe and Brown 1997). A global inhibi-tion of translation would significantly contribute to theobserved metabolic depression in torpor. All hepaticpolysome profiles from normometabolic hamsters dis-played a pronounced peak in the polysome-containingpart of the sucrose gradients, which is consistent withactive translation. Profiles from torpid animals show adisaggregation of polysomes indicated by reduced RNAcontent of fractions at the bottom of the sucrose gradi-ents.

The liver samples from torpid animals were takenwhen the minimum metabolic rate was first attained. Aswas mentioned before, at the time of entrance into tor-por at an ambient temperature of 15 �C, body temper-ature was 23.2 �C (Heldmaier et al. 1999). At thistemperature, the disaggregation of polysomes alreadyoccurs, maybe due to inhibition of translational initia-tion.

Fig. 3a–d Analysis of polysomeprofiles of liver tissue fromnormometabolic and torpidhamsters. a Ethidium bromide-stained gels and the position of18S and 28S rRNA. Numberedlanes contain isolated RNAfrom each fraction across the20–60% sucrose gradient.b Phosphorimages of Northernblots following hybridization toa 32P-labeled actin DNA probe.c Distribution of RNAthroughout sucrose gradients.Densitometrically quantifiedtotal RNA in the ethidiumbromide-stained Northern gelsof normometabolic (n=4) andtorpid (n=4) hamsters. dDistribution of actin mRNAthroughout sucrose gradients.Densitometrically quantifiedactin mRNA signals onNorthern blots ofnormometabolic (n=4) andtorpid (n=4) hamsters. Resultsrepresent means±SEM of thepercentages in every particularfraction of total RNA (c) ortotal actin signal (d) for theentire gradient

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Polysome profile analysis of liver tissue from hiber-nating golden-mantled ground squirrels revealed athreshold body temperature of 18 �C, at which transla-tional initiation is inhibited and polysomes begin todisaggregate (van Breukelen and Martin 2001). In thepresent study the distribution of total RNA isolatedfrom the fractionated gradient on agarose gel indicates adisaggregation of polysomes during entrance into torporwhen hamsters reach minimum metabolic rate at a bodytemperature of 23.2 �C. This suggests that down-regu-lation of translation occurs at an early stage of dailytorpor when body temperature is still clearly above18 �C. This may be characteristic of daily torpor wherethe development of hypothermia is not that severecompared to hibernation.

In agreement with the analysis of total RNA, amajor portion of actin mRNA was found in monoso-mal fractions, indicating a decrease in actin synthesis intorpor. However, a large portion of actin transcriptswere still found in polysomal fractions. Most likely,transcripts initiated in the normometabolic state arestill elongated in the early phase of torpor, albeit at areduced rate.

One possible mechanism for a global down-regula-tion of translation at the level of initiation is provided byphosphorylation inactivation of the eukaryotic initiationfactor 2a (eIF2a). The phosphorylation of eIF2a causesa stable binding of the recycling protein, eIF2B, pre-venting the activation of the eIF2a by GTP binding, anecessary step for translational initiation. Phosphoryla-tion of eIF2a has been shown to increase from less than2% to 13% in brains of torpid ground squirrels (Fre-richs et al. 1998). This level of phosphorylation has beenassociated with substantial decrease in translation initi-ation (Barber et al. 1995), as the stable binding of therecycling protein eIF2B to phosphorylated eIF2a givesthe limit for initiation. However, this may vary amongtissues (Oldfield et al. 1994). Other mechanisms oftranslational control may also be involved, includingmechanisms regulating translational elongation.

Acknowledgements We would like to thank F. van Breukelen forproviding detailed protocols for run-on assays as well as forpolysome profiles and for technical support via email. All experi-ments were performed according to the German animal welfarelegislation. This work was supported by grants of the DeutscheForschungsgemeinschaft.

References

Adams MP, Goodman JI (1976) RNA synthesis and RNA poly-merase activity in hepatic nuclei isolated from rats fed thecarcinogen 2-acetylaminofluorene. Biochem Biophys ResCommun 68:850–857

Barber GN, Jagus R, Meurs EF, Hovanessian AG, Katze MG(1995) Molecular mechanisms responsible for malignant trans-formation by regulatory and catalytic domain variants of theinterferon-induced enzyme RNA-dependent protein kinase.J Biol Chem 270:17423–17428

Capparelli R, Cottone C, D’Apice L, Viscardi M, Colantonio L,Lucretti S, Iannelli D (1997) DNA content differences in

laboratory mouse strains determined by flow cytometry.Cytometry 29:261–266

Frerichs KU, Smith CB, Brenner M, DeGracia DJ, Krause GS,Marrone L, Dever TE, Hallenbeck JM (1998) Suppression ofprotein synthesis in brain during hibernation involves inhibitionof protein initiation and elongation. Proc Natl Acad Sci USA95:14511–14516

Geiser F (1988) Reduction of metabolism during hibernation anddaily torpor in mammals and birds: temperature effect orphysiological inhibition? J Comp Physiol 158:25–37

Gulevsky AK, Grischenko VI, Zagnoiko VI, Shchenyavsky II, Il-yasova EN (1992) Peculiarities of functioning of protein-syn-thesizing apparatus of the hibernator (Citellus undulatus).Cryobiology 29:679–684

Heldmaier G, Ruf TP (1992) Body temperature and metabolic rateduring natural hypothermia in endotherms. J Comp Physiol B162:696–706

Heldmaier G, Steinlechner S (1981) Seasonal pattern and energeticsof short daily torpor in the Djungarian hamster, Phodopussungorus. Oecologia 48:265–270

Heldmaier G, Klaus S, Wiesinger H, Friedrichs U, Wenzel M(1989a) Cold acclimation and thermogenesis. In: Malan A,Canguilhem B (eds) Living in the cold II. John Libbey Eurotext,London, pp 347–358

Heldmaier G, Steinlechner S, Ruf TP, Wiesinger H, Klingenspor M(1989b) Photoperiod and thermoregulation in vertebrates: bodytemperature rhythms and thermogenic acclimation. J BiolRhythms 4:251–265

Heldmaier G, Klingenspor M (2002) Role of photoperiod duringseasonal acclimation in winter-active small mammals. In:Heldmaier G, Werner D (eds) Environmental signal processingand adaptation. Springer, Berlin Heidelberg New York, pp251–279

Heldmaier G, Klingenspor M, Werneyer M, Lampi BJ, Brooks SP,Storey KB (1999) Metabolic adjustments during daily torpor inthe Djungarian hamster. Am J Physiol 276:E896–906

Knight JE, Narus EN, Martin SL, Jacobson A, Barnes BM, BoyerBB (2000) mRNA stability and polysome loss in hibernatingArctic ground squirrels (Spermophilus parryii). Mol Cell Biol20(17):6374–6379

Malan A (1993) Temperature regulation, enzyme kinetics, andmetabolic depression in mammalian hibernation. In: Carey C,Florant GL, Wunder BA, Horwitz BA (eds) Life in the cold.Westview Press, Boulder, pp 242–252

Oldfield S, Jones BL, Tanton D, Proud CG (1994) Use of mono-clonal antibodies to study the structure and function ofeukaryotic protein synthesis initiation factor eIF-2B. Eur JBiochem 221:399–410

Rolfe DF, Brown GC (1997) Cellular energy utilization andmolecular origin of standard metabolic rate in mammals.Physiol Rev 77:731–758

Snyder GK, Nestler JR (1990) Relationships between body tem-perature, thermal conductance, Q10 and energy metabolismduring daily torpor and hibernation in rodents. J Comp PhysiolB 159:667–675

Storey KB (1997) Metabolic regulation in mammalian hibernation:enzyme and protein adaptations. Comp Biochem Physiol APhysiol 118:1115–1124

Storey KB, Storey JM (1990) Metabolic rate depression and bio-chemical adaptation in anaerobiosis, hibernation and estiva-tion. Q Rev Biol 65:145–174

van Breukelen F, Martin SL (2001) Translational initiation isuncoupled from elongation at 18 �C during mammalian hiber-nation. Am J Physiol Regul Integr Comp Physiol 281:R1374–R1379

van Breukelen F, Martin SL (2002) Reversible depression oftranscription during hibernation. J Comp Physiol B 172:355–361

Weber J, Jelinek W, Darnell JE Jr (1977) The definition of a largeviral transcription unit late in Ad2 infection of HeLa cells:mapping of nascent RNA molecules labeled in isolated nuclei.Cell 10:611–616

501

Whitten BK, Klain GJ (1968) Protein metabolism in hepatic tissueof hibernating and arousing ground squirrels. Am J Physiol214:1360–1362

Yamamoto M, Seifart KH (1977) Synthesis of ribosomal 5S RNAby isolated nuclei from HeLa cells in vitro. Biochemistry16:3201–3209

Zhegunov GF, Dzhordan M, Vong L (1992) Protein synthesisduring hyperthermia in ground squirrels. Biokhimiia 57:1177–1180

502