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Changes in interscapular brown adipose tissue of the rat during perinatal and early postnatal development and after cold acclimation—I. Activities of some respiratory enzymes in the tissue
Comp. Biochem. Physiol., 1970, Vol. 33, pp. 499 to 508. Pergamon Press. Printed in Great Britain
C H A N G E S IN I N T E R S C A P U L A R BROWN A D I P O S E T I S S U E OF T H E RAT D U R I N G P E R I N A T A L A N D EARLY P O S T N A T A L D E V E L O P M E N T A N D A F T E R COLD A C C L I M A T I O N - - I . A C T I V I T I E S OF SOME RESPIRATORY E N Z Y M E S IN T H E T I S S U E TUDOR BARNARD, JOSEF SKALA* and OLOV LINDBERG University of Stockholm, The Wenner-Gren Institute, Stockholm, Sweden (Received 21 J u l y 1969)
A b s t r a c t - - 1 . Specific activities of succinic dehydrogenase, ot-glycerophosphate
dehydrogenase and cytochrome oxidase were measured in homogenates of interscapular brown adipose tissue from developing and cold-acclimated rats. These activities changed in similar patterns, reaching maximal levels at 17 days post partum and after cold acclimation. 2. The total respiratory capacity of interscapular brown adipose tissue, related to body weight, was calculated for each enzyme and stage studied. They were all greatest at 5 days post partum and after cold acclimation. 3. The results are interpreted to imply that brown adipose tissue has maximal in vitro functional capacity around the fifth day of postnatal life and after cold acclimation. INTRODUCTION THE DEVELOPMENT of thermoregulation in mammals is the sum of a n u m b e r of
different processes, such as non-shivering thermogenesis, vascular regulation of heat flow, insulation, shivering thermogenesis and basal metabolism, each having an individual ontogenetic pattern. T h e functional importance of non-shivering thermogenesis may be ontogenetically localized to the early postnatal stage, at which time most thermoregulatory mechanisms are poorly developed and there is a relatively high heat loss (Hissa, 1968). In the rat, non-shivering thermogenesis has been considered to be fully developed by the eighteenth day of postnatal life, whereas the mechanisms regulating heat loss continue to develop up to about the thirtieth day post partum (p.p.) (Hahn & Koldovsk~, 1966). Non-shivering thermogenesis is also important during arousal from hibernation (Joel et al., 1964) and on cold acclimation (Smith & Roberts, 1964). Brown adipose tissue is the only organ known, the main physiological role of which is considered to be thermogenesis. Numerous studies have shown that there is a positive correlation between the amount of brown fat and capacity for * Faculty of Pediatrics, Charles University in Prague. Present address: Faculty of Medicine, Department of Obstetrics and Gynaecology, Vancouver General Hospital, Vancouver, B.C., Canada. 499
500
TUDOR BARNARD,JOSEF SKALA AND OLOV LINDBERG
non-shivering thermogenesis in an animal. T h e relative contribution of this tissue to total non-shivering thermogenesis is, however, still a matter of discussion. T h u s Imai et al. (1968) and Jansk~ & Hart (1968) postulated only a minor part of non-shivering thermogenesis to be localized within brown fat of adult rats. However, Hull & Segall (1965) and Heim & Hull (1966a, b) calculated that about two-thirds of non-shivering heat production in neonatal rabbits took place within brown adipose tissue. Jansk~ & Vot~pkovfi (1969) have concluded that the relative contribution by brown adipose tissue to total non-shivering thermogenesis is to a large extent dependent upon the age of the experimental animal. For instance, they calculated that interscapular brown adipose tissue has sufficient capacity to account for all non-shivering thermogenesis in rats only during the first 3 days of postnatal life, whereas at later stages it can be responsible for maximally only three-quarters of total non-shivering thermogenesis. Although all these conflicting results do not permit a localization of the in vivo functionally most important period for brown adipose tissue, they do support the prediction of Hahn & Koldovsk~ that the importance of non-shivering thermogenesis (and hence the role of brown adipose tissue) varies during development (see also Sk~la & Lindberg, 1969). T h e energy requirements of a tissue for synthetic or other forms of work is considered to be a major factor controlling the rate of respiration. However, in brown adipose tissue, with its thermogenic function, this situation does not apply: in this case, "the control of cellular respiration is only at the level of the mitochondrial respiratory chain" (Prusiner & Poe, 1968). So, for this organ, it seems valid to use the maximal activities of electron transport enzymes as indices of the theoretical maximal metabolic capacity of the tissue, as originally proposed by Jansk3~ (1961a, b). Therefore, the changes in the maximal activities of respiratory chain enzymes in brown adipose tissue during ontogenesis and after cold acclimation may serve to indicate the period(s) during which this tissue is functionally most important to the animal. MATERIALS AND METHODS Animals
Sprague-Dawley rats (Rattus rattus) of known ages were obtained from Anticimex, Viby, Sweden. Cold acclimation was achieved by subjecting rats of starting age 40-50 days, housed in separate cages, to an ambient temperature of 4 _+1°C for 8 weeks. Fresh weights
Interscapular brown adipose tissue (ISBAT) was removed immediately after decapitation of rats, and tissue from all animals in the same age group was placed in a tared beaker containing ice-cold 0'25 M sucrose. Tissue from 5-day-old animals and all stages thereafter was first carefully trimmed of extraneous tissues on filter paper moistened with cold 0-25 M sucrose before being blotted dry and transferred to the appropriate beaker. The pooled sample, sucrose and beaker were then weighed. The average number of animals comprising a group for each experimental stage is shown in Table 1.
501
INTERSCAPULAR BROWN ADIPOSE TISSUE OF THE R A T - - I . T A B L E 1 - - G E N E R A L CHANGES OF I S B A T
D U R I N G DEVELOPMENT AND AFTER COLD
ACCLIMATION OF RATS~ AND SAMPLE SIZES USED
Age (days)
Average body wt. (g)
1 a.p. 5.2 1 p.p. 6-0 5 p.p. 11.7 17 p.p. 36.6 30 p.p. 85.5 Cold acclimated 242.0
Average ISBAT wt. (mg)
Ratio ISBAT wt./body wt. x 103
Protein in wet weight of ISBAT* (%)
30.0 47.1 74.3 134.0 164.0
5.77 7.85 6.35 3.67 1.92
9.55 + 0.82 10-5 + 0.41 11.2 13"0 + 0.31 17.3 + 2.48
5-8 5-8 5-8 5-8 4
697"0
2"88
20"3 + 2"1
5
No. of observations
Average No. of animals for 1 pooled sample 80 50 30 20 12 2
* X + S.E. (sNn). Preparation of homogenate After weighing, the pooled tissue was chopped into small pieces with scissors and washed several times with chilled 0"25 M sucrose. Tissue was then homogenized in about 8 vol. of ice-cold 0"25 M sucrose using a glass homogenizer with a Teflon pestle rotating at approximately 800 rev/min. The homogenate was filtered through nylon in order to obtain more homogeneous samples for the enzymatic assays. Filtration was, however, omitted during preparation of the homogenates used to measure only protein concentrations in the tissue. Assays SDH (succinate: tetrazolium oxidoreductase, E.C. 1.3.99.1), ~-GPD (mitochondrial alpha-glycerophosphate : tetrazolium oxidoreductase, E.C. 1.1.95.5) and COX (cytochromec: O3 oxidoreductase, E.C. 1.9.3.1) were followed as described in Sk~la et al. (1969). In typical assays, amounts of homogenate protein used were, respectively, 0"1, 0"1 and 0"5 mg. Protein was determined by the method of Lowry et al. (1951). RESULTS It has previously been shown that the interscapular pad is quantitatively the most important site of brown adipose tissue in the rat during development and cold acclimation (Cameron & Smith, 1964; Jansk~7 & Vot~pkov~, 1969). Furthermore, the brown adipose tissue from this site is also the easiest to quickly remove, a factor of some importance when up to 150 animals were slaughtered per experiment. Therefore, the following results are based only upon an analysis of I S B A T , a point to be observed especially when comparing the results of brown fat weights presented here with those in the literature. However, ultrastructural observations have not shown any obvious differences between the perinatal development of dorsal cervical and interscapular brown fat (Bamard, 1969), so it is probable that our results are representative for rat brown adipose tissue as an organ. During development, I S B A T changed not only in size but also in colour, and therefore the ease with which it could be distinguished from the surrounding
502
TUDOR BARNARD, JOSEF SI~LAA N D OLOVLINDBERG
white adipose tissue also changed. In newborn and in cold-acclimated animals, the difference in colour between the two sorts of adipose tissue was greatest and isolation of uncontaminated pads of ISBAT was performed without difficulty. In animals at 1 day ante partum (a.p.), the tissue around the pad had not yet assumed the appearance of mature white fat and it was not easy to exactly distinguish the boundary between the brown adipose tissue and its surroundings. This difficulty was reflected in a relatively wide spread of the results for the mean weights of pooled tissue at this stage. Between 2 or 3 days after birth until at least 30 days p.p. there were increasing amounts of white adipose tissue surrounding the brown fat pad. Also, the colour of ISBAT gradually became paler, because of the increasing amounts of lipid accumulating within it (see Figs. 1-4). As shown in Table 1, the increase in weight of ISBAT was most rapid during the early stages investigated, being linear up to about 5 days p.p. and then levelling off somewhat by 30 days after birth. The weight of the tissue after cold acclimation was four times that in 30-day-old animals raised at normal ambient temperature. The variation between the individual values for any postnatal stage were within + 5 per cent of the mean. The mean total body weights during development of this strain of rats are also given in Table 1, as well as the ratio between the weight of ISBAT to that of the whole body. Unlike the growth of ISBAT, total body weight increased more rapidly between 17 and 30 days p.p. than just after birth; the ratio of these parameters therefore peaked sharply at 1 day p.p. and declined thereafter. In cold-acclimated rats there was, however, a reversal of this trend, indicating that growth of the interscapular tissue was more strongly stimulated by cold than growth of the whole animal. From these results alone, brown adipose tissue would appear to be physiologically most important during the neonatal period, but such a conclusion would ignore possible alterations in tissue composition during development. Changes in lipid content, water content and dry weight during ontogenesis of ISBAT have previously been described (Hahn et al., 1965). Measurements of tissue protein concentration were made here on all samples to enable later calculation of total enzyme activities. The results of these determinations, also shown in Table 1, showed a continuous increase in the percentage tissue protein per net weight during development, which was in agreement with the results of Hahn et al. (1965). We did not investigate to what extent the further increase in tissue protein seen in cold-acclimated rats was caused by cold acclimation, as opposed to difference in age, from the 30-day-old animals. As can be seen from Table 2, the specific activities of SDH, ~-GPD and COX were all considerably higher in homogenates of brown adipose tissue from 17-dayold rats than in adult rat liver. During development of ISBAT, the patterns described by the changes in specific activity of each enzyme were very similar. From 1 day a.p. to 5 days p.p. the specific activities increased rapidly, with a further slower increase to 17 days after birth. By 30 days p.p. all three activities had declined considerably. Some experiments using 40- and 90-day-old rats
FIGS. l-4. ISBAT of the rat during development and after cold acclimation. ( x 8,000.) Notice changes in the amount of triglycerides (T) and the locularity of the adipocytes. Mitochondria (M) appear to be less abundant at 30 days p.p. than at other stages. Other abbrelriations: glycogen (G), capillary(C). Fig. 1: 1 day a.p. ; Fig. 2 : 5 days p.p. ; Fig. 3 : 30 days p.p. ; Fig. 4: cold acclimated.
503
INTERSCAUPLAR BROWN ADIPOSE TISSUE OF THE R A T - - I .
also showed lowered activities of S D H , so this may exclude the possibility that the fall in activities was only temporary. After cold acclimation, the specific activities of all three enzymes were increased to about the same levels as were attained at 17 days p.p. TABLE 2--SPECIFIC
Age (days) 1 a.p. 1 p.p. 5 p.p. 17 p.p. 30 p.p. Cold acclimated Adult rat liver X_+ S.E.
ACTIVITIES OF
SDH,
,x-GPD
AND C O X
IN I S B A T
HOMOGENATES
SDH (/~moles succinate oxidized/min per mg protein)
a-GPD (/zmoles ~-GP oxidized/min per mg protein)
COX (/zatoms O~/min per mg protein)
0.123 + 0"0059 0.142 +_0.083 0.195 + 0.0114 0-237 + 0.0301 0.186 0'220 + 0"0143 0"155
0.072 + 0.0016 0.092 + 0.0193 0"167 + 0.0141 0"203 + 0.0072 0.134 0-186 _+0.0278 0"020
0'374 + 0.002 0.424 + 0-010 0.533 _+0-008 0-659 + 0.012 0"545 _+0.003 0"733 + 0"027 0"222
(sA/n).
Table 3 presents enzyme activities expressed per g tissue fresh weight, recalculated from specific activities, and using for every age group the average protein concentration in I S B A T . On this basis, increases in enzyme activities between 1 day a.p. and 17 days p.p. were more pronounced and no decline in activity appeared between 17 days and 30 days p.p. These differences were caused by the continually increasing concentration of tissue protein during the whole period. T A B L E 3 - - A C T I V I T I E S OF
AND C O X
PER g I S B A T
SDH a-GPD (/zmoles succinate (/zmolesct-GP COX (/~atoms oxidized/min per g) oxidized/rain per g) O2/min per g)
Age (days) 1 a.p. 1 p.p. 5 p.p. 17 p.p. Cold acclimated X~_+S.E.
SDH, a-GPD
11"8+0'56 14'9 + 0"87 21 "8 +_1"28 32"0 44"8 _+2"91
6"90+0"15 9"62 + 2.03 18'8 +_1"58 23-1 37"9 _+5"65
35'7+0"21 44'5 + 1'03 59"7 + 0'87 94"1 +0'53 149 _+5"50
(sNn).
F r o m these results, it can be concluded that development of brown adipose tissue involves not only an increase in tissue mass but also changes in activity of functionally important enzymes. Estimates of the maximal total activities in I S B A T of S D H , a - G P D and C O X were calculated by multiplying the enzymatic activities per g of tissue by the
504
TUDOR
B A R N A R D , J O S E E SK/~LA A N D O L O V L I N D B E R G
average fresh weight of the tissue, for each stage. T h e s e results are shown in T a b l e 4. T h e r e were rapid increases up to 17 days p.p. and these were followed by continued, slower rises to 30 days after birth. Because of the considerably greater fresh weight of I S B A T in cold acclimated animals, the total enzyme activities were far higher at this stage than for any of the others. TABLE 4 Age (days)
CALCULATEDTOTALACTIVITIESOF SDH, a-GPD AND COX IN ISBAT
SDH (/*moles succinate a-GPD (/*moles a-GP oxidized/min per ISBAT) oxidized/min per ISBAT)
1 a.p. 1 p.p. 5 p.p. 17 p.p. 30 p.p. Cold acclimated
0"353 0'702 1 "62 4-16 5'24 31 '0
COX (/zatoms O~/min per ISBAT)
0"205 0"453 1"39 3"56 3"78 26.4
1'07 2"10 4"44 11"6 15-4 104'0
T h e changes in total activities were related to whole body weight, using the average body weight for each stage. T h i s relationship is t e r m e d the total respiratory capacity per body weight in I S B A T , for each enzyme measured. T h e changes of these parameters, shown in Fig. 5, resembled once again the pattern of alterations of enzyme specific activities. I n this case, the peak during early development
/
0.4
0-3 x O c.)
f
/
-- 0.2
/
\
a n (.9 I
0.1 x"
0.2
tm co
0.1
iI~
l
I
I
B
5
17
30
Age,
\\
I C.A.
days
FIG. 5. Total respiratory capacity per body weight in ISBAT of some electron transport enzymes during development (B = birth) and after cold acclimation (C.A.). . , COX (/*atoms Oz/min per ISBAT per g body wt.); O SDH (/*moles succinate oxidized/min per ISBAT per g body wt.); O, ~-GPD (/*moles ~-GP oxidized/min per ISBAT per g body wt.).
I N T E R S C A P U L A R B R O W N A D I P O S E T I S S U E OF T H E R A T - - I .
505
occurred somewhat earlier, at 5 days after birth; while the value after cold adaptation was the same as the maximal value during early development. In conclusion, the specific activities of at least three electron transport enzymes in ISBAT attained their highest values, which were also similar, at 17 days after birth and after cold acclimation. In relation to body weight, ISBAT had highest total activities of electron transport enzymes at 5 days after birth and after cold acclimation, at which stages these values were the same and probably maximal. DISCUSSION
1. The significance of SDH, ~-GPD and COX activities for tissue respiratory activity Thermogenesis in brown adipose tissue, as in every other tissue in metabolic steady state, can be measured in terms of oxygen consumption independently of the nature of the mechanism involved in the oxidation process. As electron transport along the mitochondrial respiratory chain during maximal activity of brown adipose tissue seems not to be rate-limited by coupled phosphorylation (Prusiner et al., 1968; Prusiner & Poe, 1968), a study of the activity of the respiratory chain during ontogenesis and after cold acclimation of ISBAT might indicate the period(s) of functional importance of this tissue. Because both succinate and a-glycerophosphate have been shown to be excellent substrates for respiration of isolated brown fat mitochondria (Hittelman & Lindberg, 1969; Z. Drahota, personal communication), the dehydrogenases of these compounds were chosen for assay. By measurement of the enzymes as tetrazolium reductases, using the artificial electron carrier phenazine metasulphate, the involvement of a rate-limiting factor other than the amount and activity of the enzyme concerned was probably eliminated (Singer, 1966). Cytochrome-c oxidase has previously been used for calculations of the theoretical maximal contribution of brown adipose tissue to non-shivering thermogenesis (Jansk)7 & Vot~pkov5, 1969). Furthermore, comparisons with other tissues are most easily made using this enzyme. As with the dehydrogenases, the most active artificial electron carrier described for cytochrome-c, that is, T M P D (N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride), was used for this assay. The possibility of misinterpreting the results, for instance because of an extraneous rate-limiting step in an assay system, should be reduced by comparisons between the patterns of activities of the different enzymes. If metabolic control of, say, S D H occurred one might expect to see a noticeable difference between the results for this enzyme and the resuks for the others. However, since the patterns of specific activity changes for all three enzymes were very similar, it seems that no such extraneous rate-limiting steps were present. Therefore the results may be considered to represent the maximal possible respiratory activity of the tissue at the different developmental stages. Of course, in an integrated system, it may be expected that factors other than the activities of these enzymes are rate-limiting, so that the in vivo respiration of the tissue need not actually be maximal at the periods of greatest in vitro activities of the enzymes.
506
TUDOR BARNARD,JOSEF SK2~LAAND OLOV LINDBERG
2. Changes in respiratory activity of ISBAT during early ontogenesis The measured specific activities of SDH, ~-GPD and COX were all greatest at 17 days p.p. during early development (Table 2). Using the argument presented in the first part of the Discussion, it would therefore appear that the in vitro respiratory activity of the tissue is greatest at this time. In comparison, the total respiratory capacity of ISBAT (Table 4), when related to body weight, showed a slightly different pattern (Fig. 5). These results were a combination of two different processes, an alteration in the ratio of fresh weight to body weight and of changes in the respiratory capacity of the tissue. From 1 day a.p. to 1 day p.p., both these factors were increasing and hence there was also a rapid increase in the total respiratory capacity per body weight. From 1 day p.p. to 17 days p.p., whereas the ratio of fresh weight to body weight declined, respiratory capacity continued to increase. These opposite tendencies resulted in a peak of total respiratory capacity per g body weight at 5 days after birth. Finally, in the period from 17 to 30 days p.p., both fresh weight to body weight and respiratory capacity decreased, and so there was a fall in the total respiratory capacity per body weight. Thus in the rat, the period during which the total respiratory capacity of ISBAT per body weight was highest during development, coincides with that stage of the life cycle during which chemically produced heat has been supposed to be relatively most important (Hahn & Koldovsk~, 1966). Furthermore, the observed changes in total respiratory capacity per body weight support the conclusions of Jansk37 & Vot~pkov~i (1969) that considerable functional changes occur in brown fat and that the functional importance of brown adipose tissue varies during development. They calculated from the in vitro respiratory capacity of ISBAT that it could account for all non-shivering thermogenesis during the first 3 days of postnatal life. The changes in the total respiratory capacity of the tissue relative to body weight presented here indicate that the functional importance of brown adipose tissue in the rat probably extends beyond the fifth day of postnatal life.
3. Changes in respiratory activity of ISBAT on cold acclimation Exposure of rats for 8 weeks to 4°C is currently accepted as sufficient to achieve full cold acclimation. As shown in Table 2, the specific activities of SDH, ~-GPD and COX in ISBAT after cold acclimation reached approximately the same values as were maximal during early postnatal development, that is, at 17 days p.p. The similarity between these two results suggests that the maximal possible respiratory activity of ISBAT can vary up to a limit. The total respiratory capacity of ISBAT relative to body weight was also increased by cold acclimation. This was the result of increases of both respiratory activity, and of ISBAT weight relative to body weight (see also Roberts & Smith, 1967). The level of total respiratory capacity per body weight reached the same level after cold acclimation as was maximal during early postnatal development. It seems from this that the same functional capacity in ISBAT, relative to the
INTERSCAPULAR BROWN ADIPOSE TISSUE OF THE R A T - - I .
507
weight of the whole animal, is evoked during early development and after cold acclimation, in spite of the very large differences between the thermoregulatory status of rats under these two conditions. SUMMARY 1. Activities of S D H , ~ - G P D and C O X were measured in homogenates of I S B A T during ontogenesis and after cold acclimation of rats. 2. T h e specific activities of the three enzymes showed similar changes. T h e r e were rapid increases f r o m 1 day a.p. to 5 days p.p. followed by slower rises to 17 days p.p. T h e r e a f t e r activities declined by the thirtieth day after birth. 3. After cold acclimation, specific activities of the enzymes increased up to the same values as were maximal during early postnatal development. 4. T h e s e results indicate that there is a limit to which the respiratory capacity per g of tissue can be induced to increase by functional loading. 5. T h e total respiratory capacity of each enzyme in I S B A T , related to body weight, was calculated for each stage studied. T h e s e parameters, which also showed similar patterns of change, were found to increase up to 5 days p.p. T h e decline between 5 and 30 days p.p. was m o r e pronounced after the seventeenth postnatal day. 6. After cold acclimation, total respiratory capacities, related to body weight, reached the same values as were maximal during early postnatal development. 7. T h e s e results are taken to indicate that brown adipose tissue of rats has greatest in vitro functional capacity around the fifth day of postnatal life and after cold acclimation. Acknowledgements--This work was supported by Swedish National Research Council Grant No. 2171-17 Appl. No. 7979B and Swedish Medical Research Council Grant No. B 69-13X-729-04A. We wish to thank Miss Jana ~mejkalov~ for excellent technical assistance.
REFERENCES BARNARD T. (1969) The ultrastructural differentiation of brown adipose tissue in the rat. J. Ultrastruct. Res. 29, 311-322. CAMERONI. L. ~; SMITHR. E. ( 1 9 6 4 ) Cytological response of brown fat tissue in cold-exposed rats. J. Cell Biol. 23, 89-100. HAHN P. • KOLDOVSK~ r O. (1966) Utilization of Nutrients during Postnatal Development. Pergamon Press, Oxford. HAHN P., SK~LA J., VfzEK K. & NOVAKM. (1965) Changes in the reactivity of white and brown adipose tissue in vivo and in vitro to adrenalin and noradrenalin during postnatal development of the rat. Physiologia bohemoslov. 14, 546-552. HEIM T. & HULL D. (1966a) The blood flow and oxygen consumption of brown adipose tissue in the new-born rabbit. J. Physiol. 186, 42-55. HEIM T. & HULL D. (1966b) The effect of propranalol on the calorigenic response in brown adipose tissue of new-born rabbits to catecholamines, glucagon, corticotrophin and cold exposure. ,7. Physiol. 187, 271-283. HISSA R. (1968) Postnatal development of thermoregulation in the Norwegian lemming and the golden hamster. Ann. zool. fenn. 5, 345-383. 17
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HULL D. & SEGALLM. M. (1965) The contribution of brown adipose tissue to heat production in the new-born rabbit, ft. Physiol. 181, 449-457. IMAI Y., HORWITZB. A. & SMITH R. E. (1968) Calorigenesis of brown adipose tissue in coldexposed rats. Proc. Soc. exp. Biol. Med. 127, 717-719. JANSK~ L. (1961a) Total cytochrome oxidase activity and its relation to basal and maximal metabolism. Nature, Lond. 189, 921-922. JANSK~ L. (1961b) Maximal steady-state metabolism and organ thermogenesis in mammals. Presented at a Symposium for Arctic Biology and Medicine, II, Fort Wainwright, Alaska.--Comparative Physiology of Temperature Regulation. Arctic Aeromedical Laboratory, Fort Wainwright, Alaska. JANSK';~ L. & HART J. S. (1968) Cardiac output and organ blood flow in warm- and coldacclimated rats exposed to cold. Can.ft. Physiol. Pharmac. 46, 653-659. JANSK'~ L. & VOT.~PKOVAZ. (1969) Total cytochrome oxidase activity of the brown fat and its thermogenetic significance. Physiologia bohemoslav. 18. (In press.) JOEL C. P., TREBLE D. H. & BALL E. C. (1964) On a major role for brown adipose tissue in heat production during arousal from hibernation. Fedn Proc. 23, 271. LowRY H. O., ROSEBROUGH N. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent, ft. biol. Chem. 193, 265-275. PRUSINER S. B. & POE M. (1968) Thermodynamic considerations of mammalian thermogenesis. Nature, Lond. 220, 235-237. PRUSINER S. B., CANNON B. & LINDBERG O. (1968) Oxidative metabolism in cells isolated from brown adipose tissue--I. Catecholamine and fatty acid stimulation of respiration. Eur. ft. Biochem. 6, 15-22. ROBERTS J. C. & SMITH R. E. (1967) Time-dependent responses of brown fat in cold exposed rats. Am. ft. Physiol. 212, 519-525. SINGER T. P. (1966) Flavoprotein dehydrogenases of the respiratory chain. In Comprehensive Biochemistry (Edited by FLORKIN M. and STOLZ E. H.) Vol. 14, pp. 127-198. Elsevier, Amsterdam. SKALAJ., BARNARDT. & LINDBERGO. (1969) Changes in interscapular brown adipose tissue of the rat during perinatal and early postnatal development and after cold acclimation.-II. Mitochondrial changes. Comp. Biochem. Physiol. 33, 509-528. SKALA J. & LINDBERG O. (1969) Monoamine oxidase and cytochrome oxidase activities in brown adipose tissue of the rat during development. Int. J. Biochem. SMITH R. E. & ROBERTS J. C. (1964) Thermogenesis of brown adipose tissue in coldacclimated rats. A m . J . Physiol. 206, 143-148.
Key Word Index--Brown fat; rat; cold acclimation; respiratory enzymes; succinic dehydrogenase; ~-glycerophosphate dehydrogenase; cytochrome oxidase.
C H A N G E S IN I N T E R S C A P U L A R BROWN A D I P O S E T I S S U E OF T H E RAT D U R I N G P E R I N A T A L A N D EARLY P O S T N A T A L D E V E L O P M E N T A N D A F T E R COLD A C C L I M A T I O N - - I . A C T I V I T I E S OF SOME RESPIRATORY E N Z Y M E S IN T H E T I S S U E TUDOR BARNARD, JOSEF SKALA* and OLOV LINDBERG University of Stockholm, The Wenner-Gren Institute, Stockholm, Sweden (Received 21 J u l y 1969)
A b s t r a c t - - 1 . Specific activities of succinic dehydrogenase, ot-glycerophosphate
dehydrogenase and cytochrome oxidase were measured in homogenates of interscapular brown adipose tissue from developing and cold-acclimated rats. These activities changed in similar patterns, reaching maximal levels at 17 days post partum and after cold acclimation. 2. The total respiratory capacity of interscapular brown adipose tissue, related to body weight, was calculated for each enzyme and stage studied. They were all greatest at 5 days post partum and after cold acclimation. 3. The results are interpreted to imply that brown adipose tissue has maximal in vitro functional capacity around the fifth day of postnatal life and after cold acclimation. INTRODUCTION THE DEVELOPMENT of thermoregulation in mammals is the sum of a n u m b e r of
different processes, such as non-shivering thermogenesis, vascular regulation of heat flow, insulation, shivering thermogenesis and basal metabolism, each having an individual ontogenetic pattern. T h e functional importance of non-shivering thermogenesis may be ontogenetically localized to the early postnatal stage, at which time most thermoregulatory mechanisms are poorly developed and there is a relatively high heat loss (Hissa, 1968). In the rat, non-shivering thermogenesis has been considered to be fully developed by the eighteenth day of postnatal life, whereas the mechanisms regulating heat loss continue to develop up to about the thirtieth day post partum (p.p.) (Hahn & Koldovsk~, 1966). Non-shivering thermogenesis is also important during arousal from hibernation (Joel et al., 1964) and on cold acclimation (Smith & Roberts, 1964). Brown adipose tissue is the only organ known, the main physiological role of which is considered to be thermogenesis. Numerous studies have shown that there is a positive correlation between the amount of brown fat and capacity for * Faculty of Pediatrics, Charles University in Prague. Present address: Faculty of Medicine, Department of Obstetrics and Gynaecology, Vancouver General Hospital, Vancouver, B.C., Canada. 499
500
TUDOR BARNARD,JOSEF SKALA AND OLOV LINDBERG
non-shivering thermogenesis in an animal. T h e relative contribution of this tissue to total non-shivering thermogenesis is, however, still a matter of discussion. T h u s Imai et al. (1968) and Jansk~ & Hart (1968) postulated only a minor part of non-shivering thermogenesis to be localized within brown fat of adult rats. However, Hull & Segall (1965) and Heim & Hull (1966a, b) calculated that about two-thirds of non-shivering heat production in neonatal rabbits took place within brown adipose tissue. Jansk~ & Vot~pkovfi (1969) have concluded that the relative contribution by brown adipose tissue to total non-shivering thermogenesis is to a large extent dependent upon the age of the experimental animal. For instance, they calculated that interscapular brown adipose tissue has sufficient capacity to account for all non-shivering thermogenesis in rats only during the first 3 days of postnatal life, whereas at later stages it can be responsible for maximally only three-quarters of total non-shivering thermogenesis. Although all these conflicting results do not permit a localization of the in vivo functionally most important period for brown adipose tissue, they do support the prediction of Hahn & Koldovsk~ that the importance of non-shivering thermogenesis (and hence the role of brown adipose tissue) varies during development (see also Sk~la & Lindberg, 1969). T h e energy requirements of a tissue for synthetic or other forms of work is considered to be a major factor controlling the rate of respiration. However, in brown adipose tissue, with its thermogenic function, this situation does not apply: in this case, "the control of cellular respiration is only at the level of the mitochondrial respiratory chain" (Prusiner & Poe, 1968). So, for this organ, it seems valid to use the maximal activities of electron transport enzymes as indices of the theoretical maximal metabolic capacity of the tissue, as originally proposed by Jansk3~ (1961a, b). Therefore, the changes in the maximal activities of respiratory chain enzymes in brown adipose tissue during ontogenesis and after cold acclimation may serve to indicate the period(s) during which this tissue is functionally most important to the animal. MATERIALS AND METHODS Animals
Sprague-Dawley rats (Rattus rattus) of known ages were obtained from Anticimex, Viby, Sweden. Cold acclimation was achieved by subjecting rats of starting age 40-50 days, housed in separate cages, to an ambient temperature of 4 _+1°C for 8 weeks. Fresh weights
Interscapular brown adipose tissue (ISBAT) was removed immediately after decapitation of rats, and tissue from all animals in the same age group was placed in a tared beaker containing ice-cold 0'25 M sucrose. Tissue from 5-day-old animals and all stages thereafter was first carefully trimmed of extraneous tissues on filter paper moistened with cold 0-25 M sucrose before being blotted dry and transferred to the appropriate beaker. The pooled sample, sucrose and beaker were then weighed. The average number of animals comprising a group for each experimental stage is shown in Table 1.
501
INTERSCAPULAR BROWN ADIPOSE TISSUE OF THE R A T - - I . T A B L E 1 - - G E N E R A L CHANGES OF I S B A T
D U R I N G DEVELOPMENT AND AFTER COLD
ACCLIMATION OF RATS~ AND SAMPLE SIZES USED
Age (days)
Average body wt. (g)
1 a.p. 5.2 1 p.p. 6-0 5 p.p. 11.7 17 p.p. 36.6 30 p.p. 85.5 Cold acclimated 242.0
Average ISBAT wt. (mg)
Ratio ISBAT wt./body wt. x 103
Protein in wet weight of ISBAT* (%)
30.0 47.1 74.3 134.0 164.0
5.77 7.85 6.35 3.67 1.92
9.55 + 0.82 10-5 + 0.41 11.2 13"0 + 0.31 17.3 + 2.48
5-8 5-8 5-8 5-8 4
697"0
2"88
20"3 + 2"1
5
No. of observations
Average No. of animals for 1 pooled sample 80 50 30 20 12 2
* X + S.E. (sNn). Preparation of homogenate After weighing, the pooled tissue was chopped into small pieces with scissors and washed several times with chilled 0"25 M sucrose. Tissue was then homogenized in about 8 vol. of ice-cold 0"25 M sucrose using a glass homogenizer with a Teflon pestle rotating at approximately 800 rev/min. The homogenate was filtered through nylon in order to obtain more homogeneous samples for the enzymatic assays. Filtration was, however, omitted during preparation of the homogenates used to measure only protein concentrations in the tissue. Assays SDH (succinate: tetrazolium oxidoreductase, E.C. 1.3.99.1), ~-GPD (mitochondrial alpha-glycerophosphate : tetrazolium oxidoreductase, E.C. 1.1.95.5) and COX (cytochromec: O3 oxidoreductase, E.C. 1.9.3.1) were followed as described in Sk~la et al. (1969). In typical assays, amounts of homogenate protein used were, respectively, 0"1, 0"1 and 0"5 mg. Protein was determined by the method of Lowry et al. (1951). RESULTS It has previously been shown that the interscapular pad is quantitatively the most important site of brown adipose tissue in the rat during development and cold acclimation (Cameron & Smith, 1964; Jansk~7 & Vot~pkov~, 1969). Furthermore, the brown adipose tissue from this site is also the easiest to quickly remove, a factor of some importance when up to 150 animals were slaughtered per experiment. Therefore, the following results are based only upon an analysis of I S B A T , a point to be observed especially when comparing the results of brown fat weights presented here with those in the literature. However, ultrastructural observations have not shown any obvious differences between the perinatal development of dorsal cervical and interscapular brown fat (Bamard, 1969), so it is probable that our results are representative for rat brown adipose tissue as an organ. During development, I S B A T changed not only in size but also in colour, and therefore the ease with which it could be distinguished from the surrounding
502
TUDOR BARNARD, JOSEF SI~LAA N D OLOVLINDBERG
white adipose tissue also changed. In newborn and in cold-acclimated animals, the difference in colour between the two sorts of adipose tissue was greatest and isolation of uncontaminated pads of ISBAT was performed without difficulty. In animals at 1 day ante partum (a.p.), the tissue around the pad had not yet assumed the appearance of mature white fat and it was not easy to exactly distinguish the boundary between the brown adipose tissue and its surroundings. This difficulty was reflected in a relatively wide spread of the results for the mean weights of pooled tissue at this stage. Between 2 or 3 days after birth until at least 30 days p.p. there were increasing amounts of white adipose tissue surrounding the brown fat pad. Also, the colour of ISBAT gradually became paler, because of the increasing amounts of lipid accumulating within it (see Figs. 1-4). As shown in Table 1, the increase in weight of ISBAT was most rapid during the early stages investigated, being linear up to about 5 days p.p. and then levelling off somewhat by 30 days after birth. The weight of the tissue after cold acclimation was four times that in 30-day-old animals raised at normal ambient temperature. The variation between the individual values for any postnatal stage were within + 5 per cent of the mean. The mean total body weights during development of this strain of rats are also given in Table 1, as well as the ratio between the weight of ISBAT to that of the whole body. Unlike the growth of ISBAT, total body weight increased more rapidly between 17 and 30 days p.p. than just after birth; the ratio of these parameters therefore peaked sharply at 1 day p.p. and declined thereafter. In cold-acclimated rats there was, however, a reversal of this trend, indicating that growth of the interscapular tissue was more strongly stimulated by cold than growth of the whole animal. From these results alone, brown adipose tissue would appear to be physiologically most important during the neonatal period, but such a conclusion would ignore possible alterations in tissue composition during development. Changes in lipid content, water content and dry weight during ontogenesis of ISBAT have previously been described (Hahn et al., 1965). Measurements of tissue protein concentration were made here on all samples to enable later calculation of total enzyme activities. The results of these determinations, also shown in Table 1, showed a continuous increase in the percentage tissue protein per net weight during development, which was in agreement with the results of Hahn et al. (1965). We did not investigate to what extent the further increase in tissue protein seen in cold-acclimated rats was caused by cold acclimation, as opposed to difference in age, from the 30-day-old animals. As can be seen from Table 2, the specific activities of SDH, ~-GPD and COX were all considerably higher in homogenates of brown adipose tissue from 17-dayold rats than in adult rat liver. During development of ISBAT, the patterns described by the changes in specific activity of each enzyme were very similar. From 1 day a.p. to 5 days p.p. the specific activities increased rapidly, with a further slower increase to 17 days after birth. By 30 days p.p. all three activities had declined considerably. Some experiments using 40- and 90-day-old rats
FIGS. l-4. ISBAT of the rat during development and after cold acclimation. ( x 8,000.) Notice changes in the amount of triglycerides (T) and the locularity of the adipocytes. Mitochondria (M) appear to be less abundant at 30 days p.p. than at other stages. Other abbrelriations: glycogen (G), capillary(C). Fig. 1: 1 day a.p. ; Fig. 2 : 5 days p.p. ; Fig. 3 : 30 days p.p. ; Fig. 4: cold acclimated.
503
INTERSCAUPLAR BROWN ADIPOSE TISSUE OF THE R A T - - I .
also showed lowered activities of S D H , so this may exclude the possibility that the fall in activities was only temporary. After cold acclimation, the specific activities of all three enzymes were increased to about the same levels as were attained at 17 days p.p. TABLE 2--SPECIFIC
Age (days) 1 a.p. 1 p.p. 5 p.p. 17 p.p. 30 p.p. Cold acclimated Adult rat liver X_+ S.E.
ACTIVITIES OF
SDH,
,x-GPD
AND C O X
IN I S B A T
HOMOGENATES
SDH (/~moles succinate oxidized/min per mg protein)
a-GPD (/zmoles ~-GP oxidized/min per mg protein)
COX (/zatoms O~/min per mg protein)
0.123 + 0"0059 0.142 +_0.083 0.195 + 0.0114 0-237 + 0.0301 0.186 0'220 + 0"0143 0"155
0.072 + 0.0016 0.092 + 0.0193 0"167 + 0.0141 0"203 + 0.0072 0.134 0-186 _+0.0278 0"020
0'374 + 0.002 0.424 + 0-010 0.533 _+0-008 0-659 + 0.012 0"545 _+0.003 0"733 + 0"027 0"222
(sA/n).
Table 3 presents enzyme activities expressed per g tissue fresh weight, recalculated from specific activities, and using for every age group the average protein concentration in I S B A T . On this basis, increases in enzyme activities between 1 day a.p. and 17 days p.p. were more pronounced and no decline in activity appeared between 17 days and 30 days p.p. These differences were caused by the continually increasing concentration of tissue protein during the whole period. T A B L E 3 - - A C T I V I T I E S OF
AND C O X
PER g I S B A T
SDH a-GPD (/zmoles succinate (/zmolesct-GP COX (/~atoms oxidized/min per g) oxidized/rain per g) O2/min per g)
Age (days) 1 a.p. 1 p.p. 5 p.p. 17 p.p. Cold acclimated X~_+S.E.
SDH, a-GPD
11"8+0'56 14'9 + 0"87 21 "8 +_1"28 32"0 44"8 _+2"91
6"90+0"15 9"62 + 2.03 18'8 +_1"58 23-1 37"9 _+5"65
35'7+0"21 44'5 + 1'03 59"7 + 0'87 94"1 +0'53 149 _+5"50
(sNn).
F r o m these results, it can be concluded that development of brown adipose tissue involves not only an increase in tissue mass but also changes in activity of functionally important enzymes. Estimates of the maximal total activities in I S B A T of S D H , a - G P D and C O X were calculated by multiplying the enzymatic activities per g of tissue by the
504
TUDOR
B A R N A R D , J O S E E SK/~LA A N D O L O V L I N D B E R G
average fresh weight of the tissue, for each stage. T h e s e results are shown in T a b l e 4. T h e r e were rapid increases up to 17 days p.p. and these were followed by continued, slower rises to 30 days after birth. Because of the considerably greater fresh weight of I S B A T in cold acclimated animals, the total enzyme activities were far higher at this stage than for any of the others. TABLE 4 Age (days)
CALCULATEDTOTALACTIVITIESOF SDH, a-GPD AND COX IN ISBAT
SDH (/*moles succinate a-GPD (/*moles a-GP oxidized/min per ISBAT) oxidized/min per ISBAT)
1 a.p. 1 p.p. 5 p.p. 17 p.p. 30 p.p. Cold acclimated
0"353 0'702 1 "62 4-16 5'24 31 '0
COX (/zatoms O~/min per ISBAT)
0"205 0"453 1"39 3"56 3"78 26.4
1'07 2"10 4"44 11"6 15-4 104'0
T h e changes in total activities were related to whole body weight, using the average body weight for each stage. T h i s relationship is t e r m e d the total respiratory capacity per body weight in I S B A T , for each enzyme measured. T h e changes of these parameters, shown in Fig. 5, resembled once again the pattern of alterations of enzyme specific activities. I n this case, the peak during early development
/
0.4
0-3 x O c.)
f
/
-- 0.2
/
\
a n (.9 I
0.1 x"
0.2
tm co
0.1
iI~
l
I
I
B
5
17
30
Age,
\\
I C.A.
days
FIG. 5. Total respiratory capacity per body weight in ISBAT of some electron transport enzymes during development (B = birth) and after cold acclimation (C.A.). . , COX (/*atoms Oz/min per ISBAT per g body wt.); O SDH (/*moles succinate oxidized/min per ISBAT per g body wt.); O, ~-GPD (/*moles ~-GP oxidized/min per ISBAT per g body wt.).
I N T E R S C A P U L A R B R O W N A D I P O S E T I S S U E OF T H E R A T - - I .
505
occurred somewhat earlier, at 5 days after birth; while the value after cold adaptation was the same as the maximal value during early development. In conclusion, the specific activities of at least three electron transport enzymes in ISBAT attained their highest values, which were also similar, at 17 days after birth and after cold acclimation. In relation to body weight, ISBAT had highest total activities of electron transport enzymes at 5 days after birth and after cold acclimation, at which stages these values were the same and probably maximal. DISCUSSION
1. The significance of SDH, ~-GPD and COX activities for tissue respiratory activity Thermogenesis in brown adipose tissue, as in every other tissue in metabolic steady state, can be measured in terms of oxygen consumption independently of the nature of the mechanism involved in the oxidation process. As electron transport along the mitochondrial respiratory chain during maximal activity of brown adipose tissue seems not to be rate-limited by coupled phosphorylation (Prusiner et al., 1968; Prusiner & Poe, 1968), a study of the activity of the respiratory chain during ontogenesis and after cold acclimation of ISBAT might indicate the period(s) of functional importance of this tissue. Because both succinate and a-glycerophosphate have been shown to be excellent substrates for respiration of isolated brown fat mitochondria (Hittelman & Lindberg, 1969; Z. Drahota, personal communication), the dehydrogenases of these compounds were chosen for assay. By measurement of the enzymes as tetrazolium reductases, using the artificial electron carrier phenazine metasulphate, the involvement of a rate-limiting factor other than the amount and activity of the enzyme concerned was probably eliminated (Singer, 1966). Cytochrome-c oxidase has previously been used for calculations of the theoretical maximal contribution of brown adipose tissue to non-shivering thermogenesis (Jansk)7 & Vot~pkov5, 1969). Furthermore, comparisons with other tissues are most easily made using this enzyme. As with the dehydrogenases, the most active artificial electron carrier described for cytochrome-c, that is, T M P D (N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride), was used for this assay. The possibility of misinterpreting the results, for instance because of an extraneous rate-limiting step in an assay system, should be reduced by comparisons between the patterns of activities of the different enzymes. If metabolic control of, say, S D H occurred one might expect to see a noticeable difference between the results for this enzyme and the resuks for the others. However, since the patterns of specific activity changes for all three enzymes were very similar, it seems that no such extraneous rate-limiting steps were present. Therefore the results may be considered to represent the maximal possible respiratory activity of the tissue at the different developmental stages. Of course, in an integrated system, it may be expected that factors other than the activities of these enzymes are rate-limiting, so that the in vivo respiration of the tissue need not actually be maximal at the periods of greatest in vitro activities of the enzymes.
506
TUDOR BARNARD,JOSEF SK2~LAAND OLOV LINDBERG
2. Changes in respiratory activity of ISBAT during early ontogenesis The measured specific activities of SDH, ~-GPD and COX were all greatest at 17 days p.p. during early development (Table 2). Using the argument presented in the first part of the Discussion, it would therefore appear that the in vitro respiratory activity of the tissue is greatest at this time. In comparison, the total respiratory capacity of ISBAT (Table 4), when related to body weight, showed a slightly different pattern (Fig. 5). These results were a combination of two different processes, an alteration in the ratio of fresh weight to body weight and of changes in the respiratory capacity of the tissue. From 1 day a.p. to 1 day p.p., both these factors were increasing and hence there was also a rapid increase in the total respiratory capacity per body weight. From 1 day p.p. to 17 days p.p., whereas the ratio of fresh weight to body weight declined, respiratory capacity continued to increase. These opposite tendencies resulted in a peak of total respiratory capacity per g body weight at 5 days after birth. Finally, in the period from 17 to 30 days p.p., both fresh weight to body weight and respiratory capacity decreased, and so there was a fall in the total respiratory capacity per body weight. Thus in the rat, the period during which the total respiratory capacity of ISBAT per body weight was highest during development, coincides with that stage of the life cycle during which chemically produced heat has been supposed to be relatively most important (Hahn & Koldovsk~, 1966). Furthermore, the observed changes in total respiratory capacity per body weight support the conclusions of Jansk37 & Vot~pkov~i (1969) that considerable functional changes occur in brown fat and that the functional importance of brown adipose tissue varies during development. They calculated from the in vitro respiratory capacity of ISBAT that it could account for all non-shivering thermogenesis during the first 3 days of postnatal life. The changes in the total respiratory capacity of the tissue relative to body weight presented here indicate that the functional importance of brown adipose tissue in the rat probably extends beyond the fifth day of postnatal life.
3. Changes in respiratory activity of ISBAT on cold acclimation Exposure of rats for 8 weeks to 4°C is currently accepted as sufficient to achieve full cold acclimation. As shown in Table 2, the specific activities of SDH, ~-GPD and COX in ISBAT after cold acclimation reached approximately the same values as were maximal during early postnatal development, that is, at 17 days p.p. The similarity between these two results suggests that the maximal possible respiratory activity of ISBAT can vary up to a limit. The total respiratory capacity of ISBAT relative to body weight was also increased by cold acclimation. This was the result of increases of both respiratory activity, and of ISBAT weight relative to body weight (see also Roberts & Smith, 1967). The level of total respiratory capacity per body weight reached the same level after cold acclimation as was maximal during early postnatal development. It seems from this that the same functional capacity in ISBAT, relative to the
INTERSCAPULAR BROWN ADIPOSE TISSUE OF THE R A T - - I .
507
weight of the whole animal, is evoked during early development and after cold acclimation, in spite of the very large differences between the thermoregulatory status of rats under these two conditions. SUMMARY 1. Activities of S D H , ~ - G P D and C O X were measured in homogenates of I S B A T during ontogenesis and after cold acclimation of rats. 2. T h e specific activities of the three enzymes showed similar changes. T h e r e were rapid increases f r o m 1 day a.p. to 5 days p.p. followed by slower rises to 17 days p.p. T h e r e a f t e r activities declined by the thirtieth day after birth. 3. After cold acclimation, specific activities of the enzymes increased up to the same values as were maximal during early postnatal development. 4. T h e s e results indicate that there is a limit to which the respiratory capacity per g of tissue can be induced to increase by functional loading. 5. T h e total respiratory capacity of each enzyme in I S B A T , related to body weight, was calculated for each stage studied. T h e s e parameters, which also showed similar patterns of change, were found to increase up to 5 days p.p. T h e decline between 5 and 30 days p.p. was m o r e pronounced after the seventeenth postnatal day. 6. After cold acclimation, total respiratory capacities, related to body weight, reached the same values as were maximal during early postnatal development. 7. T h e s e results are taken to indicate that brown adipose tissue of rats has greatest in vitro functional capacity around the fifth day of postnatal life and after cold acclimation. Acknowledgements--This work was supported by Swedish National Research Council Grant No. 2171-17 Appl. No. 7979B and Swedish Medical Research Council Grant No. B 69-13X-729-04A. We wish to thank Miss Jana ~mejkalov~ for excellent technical assistance.
REFERENCES BARNARD T. (1969) The ultrastructural differentiation of brown adipose tissue in the rat. J. Ultrastruct. Res. 29, 311-322. CAMERONI. L. ~; SMITHR. E. ( 1 9 6 4 ) Cytological response of brown fat tissue in cold-exposed rats. J. Cell Biol. 23, 89-100. HAHN P. • KOLDOVSK~ r O. (1966) Utilization of Nutrients during Postnatal Development. Pergamon Press, Oxford. HAHN P., SK~LA J., VfzEK K. & NOVAKM. (1965) Changes in the reactivity of white and brown adipose tissue in vivo and in vitro to adrenalin and noradrenalin during postnatal development of the rat. Physiologia bohemoslov. 14, 546-552. HEIM T. & HULL D. (1966a) The blood flow and oxygen consumption of brown adipose tissue in the new-born rabbit. J. Physiol. 186, 42-55. HEIM T. & HULL D. (1966b) The effect of propranalol on the calorigenic response in brown adipose tissue of new-born rabbits to catecholamines, glucagon, corticotrophin and cold exposure. ,7. Physiol. 187, 271-283. HISSA R. (1968) Postnatal development of thermoregulation in the Norwegian lemming and the golden hamster. Ann. zool. fenn. 5, 345-383. 17
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TUDOR BARNARD,JOSEF SKALAAND OLOV LINDBERG
HULL D. & SEGALLM. M. (1965) The contribution of brown adipose tissue to heat production in the new-born rabbit, ft. Physiol. 181, 449-457. IMAI Y., HORWITZB. A. & SMITH R. E. (1968) Calorigenesis of brown adipose tissue in coldexposed rats. Proc. Soc. exp. Biol. Med. 127, 717-719. JANSK~ L. (1961a) Total cytochrome oxidase activity and its relation to basal and maximal metabolism. Nature, Lond. 189, 921-922. JANSK~ L. (1961b) Maximal steady-state metabolism and organ thermogenesis in mammals. Presented at a Symposium for Arctic Biology and Medicine, II, Fort Wainwright, Alaska.--Comparative Physiology of Temperature Regulation. Arctic Aeromedical Laboratory, Fort Wainwright, Alaska. JANSK';~ L. & HART J. S. (1968) Cardiac output and organ blood flow in warm- and coldacclimated rats exposed to cold. Can.ft. Physiol. Pharmac. 46, 653-659. JANSK'~ L. & VOT.~PKOVAZ. (1969) Total cytochrome oxidase activity of the brown fat and its thermogenetic significance. Physiologia bohemoslav. 18. (In press.) JOEL C. P., TREBLE D. H. & BALL E. C. (1964) On a major role for brown adipose tissue in heat production during arousal from hibernation. Fedn Proc. 23, 271. LowRY H. O., ROSEBROUGH N. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent, ft. biol. Chem. 193, 265-275. PRUSINER S. B. & POE M. (1968) Thermodynamic considerations of mammalian thermogenesis. Nature, Lond. 220, 235-237. PRUSINER S. B., CANNON B. & LINDBERG O. (1968) Oxidative metabolism in cells isolated from brown adipose tissue--I. Catecholamine and fatty acid stimulation of respiration. Eur. ft. Biochem. 6, 15-22. ROBERTS J. C. & SMITH R. E. (1967) Time-dependent responses of brown fat in cold exposed rats. Am. ft. Physiol. 212, 519-525. SINGER T. P. (1966) Flavoprotein dehydrogenases of the respiratory chain. In Comprehensive Biochemistry (Edited by FLORKIN M. and STOLZ E. H.) Vol. 14, pp. 127-198. Elsevier, Amsterdam. SKALAJ., BARNARDT. & LINDBERGO. (1969) Changes in interscapular brown adipose tissue of the rat during perinatal and early postnatal development and after cold acclimation.-II. Mitochondrial changes. Comp. Biochem. Physiol. 33, 509-528. SKALA J. & LINDBERG O. (1969) Monoamine oxidase and cytochrome oxidase activities in brown adipose tissue of the rat during development. Int. J. Biochem. SMITH R. E. & ROBERTS J. C. (1964) Thermogenesis of brown adipose tissue in coldacclimated rats. A m . J . Physiol. 206, 143-148.
Key Word Index--Brown fat; rat; cold acclimation; respiratory enzymes; succinic dehydrogenase; ~-glycerophosphate dehydrogenase; cytochrome oxidase.