The role of homeoviscous adaptation in mammalian hibernation

J. therra. Biol. Vol. 7. pp. 107 to 110. 1982

0306-4565 82 020107-04S03.00 0 Copyright ~ 1982 Pergamon Press Ltd

Printed in Great Britain. All rights reserved

THE ROLE OF HOMEOVISCOUS ADAPTATION IN MAMMALIAN HIBERNATION ANDREW R. COSSIN$t and HAROLD L. WILKINSON2 tDepartment of Zoology, University of Liverpool, Liverpool L69 3BX, U.K. and ZDepartment of Physiology and Biophysics, University of Illinois, Urbana. IL 61801, U.S.A.

(Receired 4 June 1981 ; accepted in rerisedJbrm 17 Auyust 1981) Al~tract--The bulk membrane fluidity of brain synaptosomes and kidney cortex microsomes of hibernating and active mammals have been compared using the steady-state fluorescence polarization technique. No consistent differences were observed indicating that homeoviscous adaptation may not be an important strategy during hibernation.

INTRODUCTION

THE MODIFICATIONof membrane fluidity to offset the direct effects of temperature changes appears to be a widespread and important cellular mechanism of temperature adaptation (Hazel & Prosser, 1974; Cossins, 1981b). Most demonstrations of such homeoviscous responses (Sinensky, 1974) have been made by measuring the motional properties of spectroscopic probes in the membranes of thermally-acclimated poikilotherms, such as fish (Cossins, 1977) or microorganisms grown at different temperatures (Cossins, 1981b). Another situation where homeoviscous responses might have adaptive value is during the hibernation of mammals, where body temperature rapidly falls by up to 35~C. Indeed, a sizeable literature exists on changes in lipid composition of membranes during hibernation (Goldman, 1975; Aloia, 1980) and there have been several accounts of functional differences between the cellular membranes of hibernating and active mammals. For example, Goldman & Albers (1979) have recently shown using fluorescence polarization spectroscopy that the fluidity of isolated brain membranes of hibernating hamsters was somewhat greater than that of corresponding preparations from active hamsters. In this study we have also compared the fluidity of membrane preparations from the brain and kidney of hibernating and active mammals. However, our results indicate no difference in the bulk membrane fluidity of hibernating and active mammals. Some possible reasons to doubt the adaptive value of homeoviscous adaptation per se during hibernation are presented. MATERIALS AND METHODS 1.6-Diphenyi-l,3,5-bexatriene and perylene were obtained from Aldrich Chemical Co. and were "puriss" grade. All other reagents were of analytical grade. * Abbreviations used: EDTA. ethylenediaminetetracetic acid: DPH. 1.6-diphenyl-l.3.5-hexatriene. 107

Animals The ground squirrel (Citellus tridecemlineatus) and hamster (Mesocricetus auratus) were obtained from Dr J. S. Willis (University of Illinois). Hibernation was induced during October and November by placing animals in individual plastic cages containing cedar shavings for bedding, placing the cages in a cold room maintained at 5-T'C and constant dark. and providing the animals with Purina rat chow and water ad libitum. Most of the animals entered hibernation within 5-6 weeks in the cold room. Animals were not used until at least 5 days after the previous arousal, as determined by disturbance of wood shavings sprinkled on the backs of hibernating animals. The body temperature of hibernating animals was within 1.5'C of the cold-room temperature. Goldfish were obtained from a commercial source and acclimated to 5"C as described previously (Cossins, 1977). All experiments were performed in Urbana during February and March. Isolation of brain synaptosomes Brain synaptosomes of mammals and goldfish were prepared exactly as described by Cossins (1977). Isolation of hamster kidney cortex microsomes All procedures were carried out at 0--4~C. The renal cortex was homogenized in 6 vol (w/v) of cold isolation medium (0.2 M sucrose, l mM EDTA*, 30raM histidine pH 7.2 at room temperature). The homogenate was filtered through two layers of gauze and centrifuged at 1500gm, , for 10rain, The pellet was resuspended in 1 vol (w/v) 2 M sucrose, homogenized and centrifuged at 1200gm~, for 10rain. The supernatant was decanted and diluted to approx. 300 mOsm using distilled water. The membranes were then centrifuged at 35,000gm~, for 15 rain and the resulting pellet was swirled with a small volume of isolation medium to resuspend the upper pink layer. This washing procedure was repeated three times and the resulting pellet was resuspended in a small volume of isolation medium.

I08

R. Cossl•s and HAROLD L. V~'ILKINSON

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IOOO / T* K Fig. 1. Arrhenius plots of polarization for DPH in brain synaptosomes of hibernating hamster (A). active hamster (El), hibernating ground squirrel (OI, rat IO) and 5:C-acclimated goldfish (Vl.

Fluorescence polarization Synaptosomal and kidney membrane preparations were labelled with DPH as described by Cossins (1977). The A4so of the final solutions was approx. 0.10. Hamster kidney membranes were labelled with perylene by shaking with perylene-coated glass beads (200 mesh) at 37~C for 90 min (Shinitzky et al., 1971). The beads were removed by centrifugation for 5 rain at 6609 and the supernatant was centrifuged at 35,000g for 30rain. The pellet was resuspended in 0.2 ml 0.25 M sucrose, 1 mM EDTA, 30 mM histidine, pH 7.2, at room temperature. An aliquot of the microsomal preparation was treated similarly except acidwashed glass beads were used. Small aliquots of membrane suspension were placed in 2 ml 0.1 M phosphate buffer for analysis. Fluorescence polarization was measured using the T-format, photon counting fluorimeter described by Jameson et al. (1977). For DPH the excitation wavelength was 357 nm and the emission was observed through a 2 mm layer of aqueous 2 N sodium nitrite followed by a Corning CS 3-73 sharpcut glass filter. For perylene the excitation wavelength was 411 nm and the emission was observed through the N a N O : filter and a Corning CS 3-72 sharpcut glass filter. In both cases polarization values were corrected for light-scattering artefact using an identical sample but without added probe as described by Shinitzky et al. (1971). Scattered light typically comprised less than 1.5% of total detected light. Temperature of cuvette contents was measured with a precision mercury thermometer. A Lauda

Arrhenius plots of the polarization of DPH in the brain synaptosomal membranes of various mammal species and 5°C acclimated goldfish are presented in Fig. 1. The membranes of the hibernating ground squirrel and the rat, and the membranes of the a~vake and hibernating hamster were prepared and analysed simultaneously to reduce the effect of variability in preparative technique. No particular significance is attached to curvilinear plots of polarization since in the case of goldfish membranes, the calculation of a semi-empirical rotational parameter using fluorescence lifetimes resulted in linear Arrhenius plots (Cossins, 1977, 1981a). The data points for all mammalian preparations irrespective of their hibernating condition were very similar. Indeed. the values obtained for the hibernating ground squirrel and rat were almost identical, The data obtained for 5:C acclimated goldfish have been included to illustrate the values of polarization obtained with the corresponding preparation of an organism that is active at the temperatures that are characteristic of hibernating mammals. In Fig. 2 the Arrhenius plots of polarization for DPH and for perylene in kidney cortex microsomes of hibernating and active hamsters are presented. For both probes the values obtained for hibernating and active animals were almost identical.

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Fig. 2. Arrhenius plots of polarization for DPH and perylene in kidney cortex microsomes of hibernating (Q. A) and active golden hamsters (O. A).

Homeoviscous adaptation and hibernation

109

quently interpreted as bulk phase transitions of bilayer lipids which induce similar Arrhenius discontiThe experiments described here suggest that there nuities in rate functions of membranes. Charnock et is no consistent difference in the membrane fluidity of al. (1980) found discontinuities in artificial membranes brain synaptosomes or kidney cortex microsomes of prepared from heart membrane lipids of both sumawake and hibernating hamsters nor between the mer-awake and winter-hibernating ground squirrels, brain synaptosomes of hibernating and non-hibernat- at 29 and 18°C, respectively. The Arrhenius discontiing species. If the compensation of fluidity was to be a nuity of (Na ~" + K +) ATPase activity of heart memviable strategy during hibernation one would expect branes was unaffected by hibernation. More direct evidence of a structural modification to observe alterations in membrane fluidity of the same magnitude, or perhaps greater, than those ob- during hibernation has been presented by Keith et al. served in thermally acclimated goldfish and green (1975) in liver mitoehondria of hibernating ground sunfish (Cossins, 1977, 1981b) which is clearly not the squirrels. They found that the Arrhenius discontinuity case. This conclusion must be qualified to emphasize for a spin probe motional parameter found for the that in common with other spectroscopic techniques, mitochondria of active individuals was absent in fluorescence polarization provides a weighted average mitochondria of hibernating individuals. However, value of fluidity for all membrane-types in the prep- a comparison of the actual values of the empirical aration. It is thus possible that significant adjustments rotational parameters measured in each case indiof membrane fluidity may have occurred in a small cates that probe motion was very considerably proportion of membranes or in a small microenviron- reduced in hibernating animals compared to active ment within the membrane (Cossins, 1981a). Another animals, in direct contrast to what one might uncertainty is the nature of the environment sampled expect. by the fluorescence probes. Both DPH and perylene The interpretation of Arrhenius discontinuities of are thought to partition into the hydrophobic region probe motion or enzymatic activity as evidence of of the bilayer by virtue of their non-polar character- phase transitions is frought with difficulty {Schreier et istics (Cossins, 1981a; Shinitzky & Barenholz, 1978). al., 1978; Silvius et al., 1978; Silvius & McEIhaney, Furthermore, DPH appears to partition equally 1981) and evidence of a more direct nature is between coexisting gel and liquid-crystalline phases required. Several recent studies in rat and rabbit (Andrich & Vanderkooi, 1976). The values of polariz- membranes have observed bulk phase transitions by ation obtained from studies on cellular membranes differential scanning calorimetry but at temperatures are broadly similar to those obtained with phospho- at or below 0:C (reviewed in Aloia, 1980). Charnock lipid liposomes containing appropriate molar ratios et al. (1980) have also detected unequivocal thermoof cholesterol (Wagg & Cossins, unpublished obser- tropic phase transitions using calorimetric techniques vations), suggesting that membrane proteins have no but at 18-25°C in active animals and 5-16:C in hibergreat influence in this respect. nating animals. The authors calculate that only 2% or In direct contrast with the present results, Goldman less of the membrane lipids were involved in these & Albers (1979~ have observed a consistently greater transitions so the precise significance of Arrhenius fluidity in brain microsomes of hibernating hamsters discontinuities of spin probe motion and enzyme accompared to the corresponding preparations of tivity is not clear. awake hamsters. The differences observed were comThis brief review of the literature concerning paratively small with a 4-8~C shift of the Arrhenius adjustments of the physical state of membranes illusplots of microviscosity along the temperature axis for trates the confusion that exists in the occurrence of a 30-35°C difference in body temperature (an efficacy fluidity compensations during mammalian hiberof 0.1-0.25, see Cossins (1981b)). However, no correc- nation. There are additional reasons to question the tions were made for light-scattering artefacts, which at adaptive value of homeoviscous responses during hi5% of total fluorescence would have a marked effect bernation. Firstly, it is generally accepted that the upon the measured values of polarization. Charnock adaptive strategy of eurythermal fish such as goldfish et al. (1980) using electron-spin resonance spec- is compensatory, so that comparable levels of Iocotroscopy have also demonstrated a greater fluidity of motory activity, behaviour and general metabolism artificial membranes prepared from the isolated heart are possible over a wide range of seasonal temperalipids of active and hibernating ground squirrels, the tures (Prosser, 1973; Precht et al., 1973). By contrast, difference being approximately half that required to the adaptive strategy of hibernation is to reduce maintain membrane fluidity constant at hibernating energy expenditure to a minimum during periods of and normothermic temperatures. inclement weather and reduced food availability. One Other studies of possible modifications of mem- might, therefore, expect hibernating mammals to disbrane fluidity during hibernation have emphasized play physiological adaptations that permit survival at the adaptive importance of lowering the phase tran- low body temperatures {i.e. resistance adaptations~ sition temperature to prevent the induction of unfa- rather than capacity adaptations which preserve elevourable membrane states at low hibernating tem- vated activities of the various physiological processes. peratures, For example, Raison & Lyons (1971) and Thus, in general, there is no reason to expect wideMcMurchie & Raison (1975) have found that during spread compensatory cellular responses such as hibernation the Arrhenius plots of both membrane- homeoviscous adaptation. Cellular adaptations might bound enzyme activity and spin probe motion were be sought most profitably in tissues whose functions linear over the temperature range where discontinui- are maintained during hibernation, albeit at a intenties were observed in preparations from active indi- sity that is a small fraction of that in normothermic viduals. Arrhenius discontinuities of this sort were fre- individuals. Examples might include synaptic mereDISCUSSION

110

ANDREW R. COSSlNSand HAROLDL. WILKINSON

branes of brain-stem neurons and perhaps mitochondrial membranes of brown fat. Secondly, hibernating mammals often undergo transient and spontaneous arousals during the hibernating period which involves rapid changes in body temperature, a temperature excursion that is often greater than that easily tolerated by eurythermal fish (Hoyland et al., 1979). In view of the time required for the adjustment of membrane lipid composition for homeoviscous purposes it seems unlikely that membrane fluidity can be appreciably modified during these brief transitional periods. It is necessary, therefore, to decide whether it is strategically most advantageous for the animal to adjust membrane fluidity and function for hibernating or normothermic temperatures. In view of the more demanding functional requirements of normothermic mammals it would seem more reasonable to expect bulk membrane fluidity to be maintained during hibernation at the presumed optimal state for normothermic animals. The results presented here are in agreement with this prediction. Acknowledgements--A. R. C. was the recipient of a Wellcome Trust Travel Grant. This work was supported in part by Grant BMS 01587 from the National Science Foundation to Professor C. L. Prosser. We thank Professor G. Weber for use of his fluorescence instrumentation, and Dr J. S. Willis for specimens provided, for guidance and helpful discussions.

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