crease in NST capacity (Ashwell et al. 1983; Bouillaud et al. 1984; Heldmaier and Buchberger 1985; Trayhurn et al. 1987). The thermogenic activity of BAT, and ...
J Comp Physiol B (1994) 164:159-164
Joumalof
"e ComparatN
BIochemical,
Systemic,
and Envlron-
Physiology B ~;~~\Og. © Springer-Verlag 1994
Long photophase is not a sufficient stimulus to reduce thermogenic capacity in winter-acclimatized short-tailed field voles (Microtus ugrestis) during long-term cold acclimation R.M. McDevitt, J.R. Speakman Department of Zoology, University of Aberdeen, Aberdeen AB9 2TN, Scotland, UK Accepted: 11 November 1993
Abstract. The thermogenic capacity of brown adipose tissue in winter- and summer-acclimatized short-tailed field voles (Microtus agrestis) was investigated by exam ining changes in mass of brown adipose tissue, the ratio of white adipose tissue to brown adipose tissue, the con centration of the uncoupling protein (thermogenin) in whole depots (ug) and in mitochondrial mass (ugmg') and the activity of cytochrome c oxidase in the depots (mmolmin"). The concentration of thermogenin in win ter-acclimatized voles (11 = 8), per brown adipose tissue depot and per mitochondrial mass, was significantly higher than in summer-acclimatized voles (11 = 6). There was no significant difference in the level of cytochrome c oxidase activity between these two groups. Four groups of winter-acclimatized voles (11 = 6 in each group) were exposed to 5 °C for 10,20,50 and 100 days in a 14L:lOD photoperiod. Body mass, brown adipose tissue mass, white adipose tissue mass and basal metabolic rate were significantly positively related to the length of time cold exposed up to 100 days. There was a significant inverse relationship between the ratio of white to brown adipose tissue mass and the duration of cold exposure. There was no significant relationship between thermogenin concen tration, either per depot or in mitochondrial mass of brown adipose tissue, with the length of time cold ex posed. The level of cytochrome c oxidase activity in creased significantly from control levels to a maximum after 10 days in the cold but decreased from 10 days onwards. In winter-acclimatized M. agrestis, a 14L:lOD photoperiod is not a sufficient stimulus to reduce ther mogenic capacity during cold acclimation. Indeed, some changes in the indirect parameters reflecting thermogene sis, notably the increase in basal metabolic rate and the decrease in the ratio of white to brown adipose tissue Abbreviations: BAT, brown adipose tissue; BM, body mass; BMR,
basal metabolic rate; MR, metabolic rate; MSD, minimum signifi cant difference; NST, non-shivering thermogenesis; SDS, sodium dodecyl sulphate; UCP, uncoupling protein (= thermogenin); }Dz, oxygen consumption; WAT, white adipose tissue Correspondence to: R.M. McDevitt
mass, indicated that despite the long photophase the thermogenic capacity was slightly further enhanced dur ing the cold acclimation. Key words: Long photophase - Cold acclimation Brown adipose tissue - Thermogenic capacity - Field vole, Microtus agrestis
Introduction The principal response of small endothermic mammals that are active in winter to seasonally decreased ambient temperatures is to increase the capacity for heat produc tion (Pohl 1965; Jansky 1973; Rosenmann et al. 1975; Feist and Rosenmann 1976).This change in thermogenic performance is primarily achieved by an increase in the capacity for NST, which originates in BAT (Jansky 1973; Foster and Frydman 1979; Himms-Hagen 1986).The ca pacity for NST in BAT depends on the concentration of the UCP (thermogenin), which is unique to BAT (Can non et al. 1982). Hypertrophy of BAT occurs in a variety of animals (e.g. hamsters Phodopus sungorus, mice Mus sp. and ground squirrels, Spermophilus sp.) in response to a suite of environmental stimuli, including cold exposure (Cannon and Nedergaard 1983; Girardier 1983), short photophase (Heldmaier et al. 1981) and a combination of the two (Klingenspor et al. 1989). However, changes in the mass of BAT are insufficient to adequately character ise NST capacity. Changes in the ratio of BAT mass to WAT mass are a more useful morphological parameter for establishing changes in the NST capacity of BAT depots (Trayhurn and Milner 1989), with a lower WAT / BAT ratio indicating a greater NST capacity. In addi tion, increases in NST capacity during acclimatization and acclimation are often correlated with an increase in BMR. However, measurements of the concentrations of UCP, and a mitochondrial activity marker, such as cy tochrome c oxidase are necessary to fully quantify ther mogenic capacity of BAT (Trayhurn and Milner 1989).
160
R.M. McDevitt, .T.R. Speakman: Photo phase and cold acclimation in Microtus
During cold acclimation, the concentration of UCP in BAT increases significantly, in conjunction with the in crease in NST capacity (Ashwell et al. 1983; Bouillaud et al. 1984; Heldmaier and Buchberger 1985; Trayhurn et al. 1987). The thermogenic activity of BAT, and thus the capacity for NST, also increases in small free living en dothermic mammals during seasonal acclimatization (Heldmaier and Steinlechner 1981; Klaus et al. 1988). Short photophase has been generally recognised as a more reliable environmental cue than decreasing ambi ent temperature, for triggering seasonal physiological events such as reproduction, hibernation and acclima tization (Heldmaier et al. 1989) and daily rhythms such as body temperature (Ruf et al. 1987).Non-shivering thermo genesis increases significantly in rodents exposed to short photophase (Haim 1982; Haim and Yahav 1982; Held maier et al. 1989) and in some species the increase in NST may exceed that found during natural acclimatization (Haim and Yahav 1982).Normally, however, the response to short photophase alone is less than the effects of natu ral acclimatization. Hence, Heldmaier et al. (1982) found that 50% of the seasonal increase in NST in Djungarian hamsters could be attributed to the triggering effects of short photophase and the remaining 50% was triggered by cold exposure. The increase in NST in response to short photophase, like that due to cold exposure, is a result of an increase in the concentration of UCP and respiratory enzymes [Djungarian hamsters, Heldmaier et al. (1981), wood mice Apodemus sylvaticus and bank voles Clethrionomys glareolus, Klaus et al. (1988)]. Since short photophase stimulates an increase in ther mogenic capacity in animals, even when they are not exposed to the cold, we wanted to see if long photophase would reverse this stimulatory effect in animals which were already cold acclimatized and kept in sustained cold. We investigated the effect of exposure to a long photophase during long-term cold acclimation (up to 100 days) on BAT mass, concentration of UCP (per BAT depot and in BAT mitochondrial mass), the concentra tion of cytochrome c oxidase and BMR in winter-accli matized short-tailed field voles (Microtus agrestis). Materials and methods Study animals. A total of 38 short-tailed field voles were live-trapped in the north-east of Scotland (57 ON) in July and November 1992. The voles caught in November (II = 32) were randomly divided into one control (n = 8) and four experimental groups (n = 6). The length of time the groups were cold exposed varied; control voles were not cold exposed, the other groups were exposed for either 10, 20,50 or 100 days at 5 "C. The voles caught in .Tuly (II = 6) were not cold exposed. These summer animals were used in an assessment of seasonal differences in thermogenic capacity and were compared directly with the winter control voles. During cold exposure voles were kept singly in rodent boxes (30 x 15 x 15 em) with food and water provided ad libitum in a constant temperature cold room (5 ± 1.0 "C) and a 14L:l0D photoperiod. We chose this photoperi od because it was the same as that experienced by these animals in the wild as they changed from winter to summer acclimatization. The exposure temperature was close to the mean daily winter tem perature at the site where the animals were captured. BM of the animals was measured approximately weekly throughout their peri od of cold exposure.
Measurement of BMR. To evaluate BMR we measured O 2 con
sumption (mlmin') using an open-circuit respirometer system which has been described in detail elsewhere (Hayes et al. 1992). Voles were denied food for 1 h prior to determinations of MR and for the duration of each experiment. Measurements were made at 25°C, the lower critical temperature of M. agrestis (Hayes et al. 1992). Each V0 2 measurement lasted for 3 h and the lowest V0 2 over a consecutive to-min period was taken as the BMR. Dissection procedure. After each group of voles had been cold ex posed for the predetermined length of time and BMR had been measured, they were killed by CO 2 overdose. The voles were dissect ed dorsally, on an insulated ice-packed tray with an aluminium surface, and the interscapular BAT was removed as rapidly as pos sible (:s: 1 minute from death) weighed (0.01 g accuracy, Oertling R20) and immediately frozen in liquid N 2 (-196°C). BAT tissue samples were kept deep frozen (-80°C) until subsequent analysis 3 months later. The carcasses were skinned and all visible subcuta neous WAT was removed, dried to a constant mass at 60°C (3 days) and reweighed (0.01 g accuracy). Determination of thermogenic capacity in BAT The concentration of UCP in the BAT whole-tissue samples was measured by im munoassay using Western blotting techniques and the level of cy tochrome c oxidase activity, per BAT depot, was assessed using a spectrophotometric assay (Trayhurn et al. 1987). Mitochondrial protein was separated by SDS gel electrophoresis, blotted onto nitrocellulose and probed with a rabbit anti-ground squirrel UCP serum. Antibodyjantigen complexes were detected with an en hanced chemi-luminescence protocol (ECL Amersham, UK) (Tray hurn et ai, 1987; Trayhurn and Milner 1989). Thermogenic capacity was therefore measured as UCP concentration per BAT depot (ug), per mitochondrial protein (ug-mg"), and the activity of cytochrome c oxidase per BAT depot (mmol-min "). Statistical analysis. The concentration of UCP and cytochrome c oxidase in summer- and winter-acclimatized voles were compared using two sample t-tests. The effect of duration of cold exposure on BAT mass, WAT mass, UCP concentration (per BAT depot and in mitochondrial mass) and cytochrome c oxidase activity were analysed by least-squares linear regression and ANOV A. Differ ences between means were compared using the MSD test (Sokal and Rohlf 1981). The influence of all the above parameters on BMR was analysed using a stepwise multiple regression with backwards deletion. There may have been a size bias introduced into the data set because only four of six voles survived the maximum 100 days in the cold and these voles included the largest from the total sam ple; therefore, BM, BAT and WAT masses were analysed both including and excluding these individuals. Continuous data on BM were available for 100-day voles and these were analysed using a repeated-measures one-way ANOVA.
Results
Seasonal differences in thermogenic capacity of BA T in Microtus agrestis Winter-acclimatized voles (control group) had con centrations of U CP per BAT depot about four times greater (124.3 ± 75.7 ug) than summer-acclimatised voles (28.5 ± 8.8 ug). These winter levels were significantly higher (t = 2.8, df = 12, P = 0.048). The concentration of UCP in BAT mitochondria of winter-acclimatized voles (11.4 ± 3.0 ugmg") was also significantly higher (df = 4, t = 3.5, P = 0.024) than in summer acclima tized animals (6.4± 1.1 ug-mg "). However, there was no significant difference in cytochrome c oxidase activity
161
R.M. McDevitt, J.R. Speakman: Photo phase and cold acclimation in Microtus
~
150
'0
I!i!I
UCP (tissue)
IITl
UCP (mitochondria)
D
Cytochrome c-oxidase
III C.
III
o o
36
*
200
I
T
30 24
-9
T
T T
