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Effects of cold acclimation and arousal from hibernation on brown fat lipid and protein in the golden hamster (mesocricetus auratus)
Comp. Biochem. Physiol., 1969, Vol. 31, pp. 111 to 119. Pergamon Press. Printed in Great Britain
EFFECTS OF COLD ACCLIMATION AND AROUSAL FROM HIBERNATION ON BROWN FAT LIPID AND PROTEIN IN THE GOLDEN HAMSTER (MESOCRICETUS A URATUS) DALE D. F E I S T and W I L B U R B. QUAY Department of Zoology, University of California, Berkeley, California
(Received 21 February 1969) A b s t r a c t - - 1 . Total lipid and protein are monitored in interscapular, cervical
and thoracic brown fat deposits from normal control, cold-acclimated, hibernating and newly aroused golden hamsters (Mesocricetus auratus). 2. During cold acclimation total lipid declines significantly while protein content increases markedly in all brown fat deposits. In contrast to interscapular and cervical deposits, thoracic brown fat contains a higher percentage of protein than lipid after cold acclimation. 3. After cold acclimation brown fat retains the same lipid and protein levels in hamsters exposed to very short and very long hibernating periods. 4. Newly aroused hamsters show little or no evidence of loss of brown fat lipid during arousal from hibernation. This contrasts with findings of other workers that ground squirrels lose a substantial percentage of brown fat lipid during arousal. INTRODUCTION MUCH evidence now indicates that brown fat plays an important role in nonshivering thermogenesis during cold acclimation and arousal from hibernation in mammals (Joel, 1965; Smalley & Dryer, 1967). Of the various substrates which may be involved in the biochemical processes of thermogenesis lipids may be significant (Masoro, 1966; Prusiner et al., 1968). Investigators have reported changes in the amount and composition of lipids in brown fat in association with cold acclimation, hibernation, and arousal from hibernation. Both cytological (Cameron & Smith, 1964) and chemical (Steiner & Cahill, 1964) studies show a decrease in the amount of lipid during cold acclimation in rats. Higher lipid levels have been reported in bat brown fat during hibernation (Wells et al., 1965). Soumalainen & Herlevi (1951 ) found a decrease in size and number of sudanophilic particles in the hedgehog brown fat immediately after arousal from hibernation. A decrease in brown fat glyceride levels was observed in the ground squirrel (Citellus lateralis) after arousal from hibernation (Spencer et al., 1966). Joel et al. (1964) and Joel (1965) have reported a loss of about 50 per cent of the lipid in brown fat of the thirteenlined ground squirrel during arousal from hibernation. J o e l concluded that complete oxidation of the lipid lost during arousal could provide more than enough calories to warm the ground squirrel to complete arousal. 111
112
DALE D. FEIST AND WILBURB. QUAY
T h e work reported here was initiated in the course of investigations of norepinephrine metabolism in b r o w n fat of hibernating and non-hibernating hamsters (Feist & Quay, 1968). I t was felt to be of value to determine what changes, if ally, in total lipids and proteins in several different brown fat deposits accompany the processes of cold acclimation and arousal f r o m hibernation in the Syrian golden hamster. MATERIALS AND M E T H O D S Adult golden hamsters (Mesocricetus auratus) were housed in individual metal cages with wood-chip bedding in either a warm room at 22 + 1°C or a cold room at 5 + 1°C. They were maintained on 8 hr of artificial illumination per day and given Purina rat chow and water adlib. The animals were subdivided as follows: (1) normal control (NC) animals maintained at 22 + 1°C ; (2) cold-acclimated (C) hamsters kept at 5 + 1°C, having hibernated, but not in hibernation at the time of killing; (3) hibernating (H) animals in deep hibernation with a rectal and/or cheek pouch temperature of 5-7°C; (4) newly aroused (A) animals which have attained a rectal temperature of 36-38°C after artificial stimulation by taking of rectal or cheek pouch temperature to arouse from deep hibernation. Rectal and cheek pouch temperatures were monitored with a small animal thermistor probe attached to a Yellow Springs Instrument Company telethermometer. Experiments were conducted between the final week in March and the first week of September. The animals were killed between 10.30 a.m. and 3.15 p.m. by rapid decapitation. Interscapular brown fat, cervical brown fat (from both sides of the ventral midline in the mid-cervical region) and thoracic brown fat (from along the course of the thoracic segment of the posterior vena cava) were excised and frozen on dry ice. The tissues were stored in a dry-ice chest at - 4 0 ° C until analysis. The analysis of total lipids was performed essentially according to Folch et al. (1957). Protein content was assayed by the method of Lowry et al. (| 951). Wet weight of the tissues was determined on a torsion balance. Lipid and protein are expressed in terms of mg/g dry wt. of tissue on the assumption that combined lipid plus protein constitute the dry weight and that carbohydrates and nucleic acids make up a negligible percentage of the total dry weight (Stair & Smit-Vis, 1966; Thomson et al., 1968). The "Student" t-test was used to estimate the statistical significance of the difference between mean values (Simpson et al., 1960).
RESULTS Body weight changes during cold exposure T h e total b o d y weights of hamsters in three different experimental sets were maintained or increased in normal control animals and decreased in all animals exposed to the cold. T h e s e results can be seen in T a b l e 1 along with descriptive data for the three experimental sets. Each experiment contains normal controls (NC), cold-acclimated (C), hibernating (H) and newly aroused (A) animals (except experiment I I which only contains hibernating and aroused animals) and is based on the length of time elapsed f r o m the onset of the hibernation period to the time of autopsy and excision of tissues. T h e length of cold exposure is m u c h shorter in experiment I than in experiment I I I . T h e length of the hibernation period is shortest in experiment I and longest in experiment I I I . (Hibernation period as defined here represents a series of entries and arousals f r o m single bouts of deep hibernation.)
Males Males Females
I II III
5~ 10 11
16 8 24
N* 106+4(16) 109 + 2 (8) 102+3(24)
All
At start C
126+13(4) 99_+3(4) --1 0 0 + 6 ( 6 ) 89_+6(6)
NC t
89+3(4) 80 + 2 (4) 81+4(6)
H
At autopsy A 85+4(4) 82 + 2 (4) 78+4(6)
(Mean ~ standard error (N*))
* N = n u m b e r of animals. t Groups : NC = normal controls, C = cold-acclimated, H = hibernating, A = newly aroused.
Sex
Experiment No.
Age at autopsy (months)
Body weights (g)
TABLE 1 - - B O D Y WEIGHTS AND EXPERIMENTAL TREATMENTS
8-10 16 16
1-2 7-9 11-14
Cold Hibernation (re: C,H,A,) (re: H,A,)
Length (weeks) of
114
DALS D. FI~ISTAND WILBUR B. QUAY
Lipid and protein changes during cold acclimation Interscapular brown fat from normal control animals in experiment I (see Fig. 1) contains about 64 per cent lipid and 36 per cent protein/dry wt. of tissue. During cold acclimation there is a significant loss ( P < 0.05) of about 23 per cent of Intecscopulor Brown f'of;
PROTEIN
LIPID
_}*
70C
*P<.05
I
(NC vsC)
60C 50C
40C .~
30c 20C
IOC
I
-
I
o
Groups: NC
H A
NCC
!i H A
Fxo. 1. Lipid and protein content of interscapular brown fat in normal (NC), cold-acclimated (C), hibernating (H) and newly aroused (A) hamsters exposed to short cold acclimation and hibernating periods. Values given = mean + S.E. of the mean. [ ~ = LIPID
[]=PROTEIN
P: (NC vs C) ,x--x--,~< .001 "~"~ < ,01
90C
(H vs A] "x'< . 0 5
.~ ,11-,)t~ 80O
70C >" 600 "o O~ 500
.... I--Z'~
r~.-" 40O 300 .
!~.-~
200 IO0
0
Groups: NC C H A Tissue:
Inlerscopulor
NC C H A Brown Fat
NC C H A
NC C H A
Cervicol Brown For
NCCHA
NCCHA
Thorocic Brown Fo!
FIc. 2. Lipid and protein content of interscapular, cervical and thoracic brown fat in normal (NC), cold-acclimated (C), hibernating (H) and newly aroused (A) hamsters exposed to long cold acclimation and hibernating periods. Values given = mean + S.E. of the mean.
4
A
Change (%) P
N 4
Group H
TABLE 2--LIPID
- 3 N.S.
546.9 + 14.2
Lipid 565.9 + 12.9
+2 N.S.
453.1 + 14.2
Protein 434.1 + 12-9
Interscapular brown fat
- 3 N.S.
524.9 + 16.5
Lipid 540.3 + 13.0
+3 N.S.
475.1 + 16.5
(H)
AND N E W L Y AROUSED
+2 N.S.
409-2 + 29.5
Lipid 400.6 + 20-1
+ 1"5 N.S.
590.8 + 29.5
Protein 599.4 + 20-1
Thoracic brown fat
FROM H I B E R N A T I N G
Protein 459.7 + 13.0
Cervical brown fat
(mg/g DRY TISSUE) I N BROWN FAT DEPOSITS (A) HAMSTERS I N M I D - H I B E R N A T I O N PERIOD
AND P R O T E I N CONCENTRATIONS
tJi
116
DALE D. FEISTANDWILBURB. QUAY
the tissue lipid and a significant increase ( P < 0.05) in protein content of about 41 per cent. Interscapular brown fat and cervical brown fat from normal control hamsters in experiment III (see Fig. 2) contains about 80 per cent lipid to 20 per cent protein/dry wt., while thoracic brown fat contains 67 per cent lipid. During cold acclimation the amount of lipid diminishes significantly (see Fig. 2) by 30-40 per cent at all brown fat sites while protein increases markedly by 86--136 per cent. In the thoracic deposit the ratio of lipid to protein was almost completely reversed during cold acclimation. Thoracic brown fat from all cold-acclimated, hibernating and newly aroused animals always contained more protein than lipid.
Lipid and protein of brown fat in hibernating and aroused hamsters In hibernating hamsters from all three experiments (I, II, III), artificial stimulation by manual determination of rectal and cheek pouch temperatures induced arousal. Arousal from a body temperature of 5-6°C to 36-37°C at an ambient temperature of 5 + I°C was completed within 3-4 hr (usually between 3 and 3.5 hr) in all animals. In all three experiments (I, II, III), interscapular brown fat and cervical brown fat from newly aroused hamsters showed no significant change in the content of lipid or protein when compared with the same tissue from hibernators (see Figs. 1, 2; Table 2). Thoracic brown fat in group II (Table 2) showed no alteration in lipid content after arousal. But thoracic brown fat in group III (Fig. 2) lost 15.5 per cent of the lipid during arousal ( P < 0.05). DISCUSSION The decline in body weight which accompanies cold exposure in these hamsters is consistent with the findings of a number of investigators (Lyman, 1948; Chaffee et al., 1964; Smit & Smit-Vis, 1966). During this same period the absolute mass of brown fat has been shown to increase (unpublished results from this laboratory; Chaffee et aL, 1964). The concomitant increase in protein and fall in lipid content in the three brown fat deposits as shown here affirms the results previously reported in hamsters (Chaffee et al., 1964) and rats (Steiner & Cahill, 1964) during cold acclimation. Biochemical investigations of synthetic and oxidative metabolic activities of brown fat during cold acclimation in rats (Smith & Roberts, 1964; Steiner & Cahill, 1964; Himms-Hagen, 1965; Roberts & Smith, 1967), hamsters (Baumber & Denyes, 1964; Chaffee et al., 1964) and ground squirrels (Chaffee et al., 1966) have provided evidence for an enhanced thermogenic capacity. Presumably the changes in lipid and protein during cold acclimation are related to this enhancement. In this study there was no difference in the percentage of lipid in brown fat deposits of cold-acclimated and hibernating hamsters killed early in the hibernating period from those killed late in the hibernating period. Smit & Smit-Vis (1966) observed histochemically only a slight decrease in lipid of interscapular brown fat in the later stages of the hibernating period of the hamster. They suggest that it is not likely that brown adipose tissue has much nutritive value during lethargy and arousal.
COLD A C C L I M A T I O N AND H I B E R N A T I O N I N T H E G O L D E N HAMSTER
117
The demonstration here that the percentage of total lipid does not decline significantly in any brown fat deposit at the end of arousal (except for a 15.5 per cent loss of lipid in thoracic brown fat in experiment III) differs from results reported in two species of ground squirrels. Spencer et al. (1966) report losses of between 25 and 38 per cent of brown fat glycerides at the end of arousal in Citellus lateralis. Joel (1965) describes a loss of about 50 per cent of the brown fat lipid in newly aroused Citellus tridecemlineatus. The lipid level was shown to return to near normal by 15--46 hr after completion of arousal. Joel also found an increase in plasma free fatty acids and glycerol during arousal. He has suggested that brown fat may perform two quantitatively important roles during arousal from hibernation: (1) oxidation of lipid to produce heat within the tissue; (2) release of large amounts of substrates into the circulation for utilization by other tissues. If brown fat in arousing hamsters performs these roles, one might expect to find a concomitant marked decrease in lipid as found in the ground squirrel. It is possible that there is a rapid loss of lipid during arousal combined with an equally rapid replacement which would resuk in no apparent loss. Another possibility is that during arousal in hamsters, thermogenesis in brown fat may result (at least in part) from internal hydrolysis and resynthesis of triglycerides with little or no loss of free fatty acids and glycerol. This would seem to be supported by evidence for accelerated triglyceride cycling--which may act as an ATPase system for heat production--in brown fat of cold-acclimated rats (Ball & Jungus, 1961; HimmsHagen, 1965) and new-born rabbits (Dawkins & Hull, 1964). Glycerol kinase, essential for re-esterification of glycerol and maintenance of triglyceride recycling, has been demonstrated in brown fat from rats and ground squirrels (Treble & Ball, 1963; Joel, 1965). It may be operative in hamster brown fat. It also seems possible that the quantitative contribution of brown fat (or brown fat lipid) to the total thermogenic response relative to the contribution of other nonshivering and shivering tissues may not be as great in the arousing hamster as in other species of hibernators. Attempts at quantitative estimation of the calorie contribution of brown fat to the total warming process based on in vitro and in vivo experiments have not yet clarified this matter (see Hayward & Lyman, 1967; Smalley & Dryer, 1967; Thomson et al., 1968). Hayward & Lyman (1967) have reported from studies of curarized and non-curarized animals that while brown fat contributes to warming, shivering is essential to normal arousal in hamsters and dormice but not in bats. They do not give any information on the importance of shivering and brown fat in ground squirrels. It would be of interest to know how much lipid is lost from bat brown fat during arousal. These possibilities remain to be tested in the arousing hamster in order to elucidate the role of brown fat and its lipid. Acknowledgements--The authors wish to express their appreciation to Dr. Glenn D. Bissel, State Health Department, Fairfield, California, for generously providing the hamsters used in this study, to Mr. William Young for animal room assistance and to Mrs. Emily Reid for final rendering of the figures.
118
DALE D. FEIST AND WILBtm B. QUAY
This work was supported in part by U.S.P.H.S. predoctoral fellowship G M 5-Fl-GM-32,590 (to D. D. F.) and research funds, University of California Berkeley (to W. B. Q.). REFERENCES BALL E. G. & JUNGUS R. (1961) On the action of hormones which accelerate the rate of oxygen consumption and fatty acid release in rat adipose tissue in vitro. Proc. hath. Acad. ScL U.S.A. 47, 932-941. BAUMBERJ. & DENYES A. (1964) A c e t a t e - l - C utilization by brown fat from hamsters in cold exposure and hibneration. Can.]. Biochem. 42, 1397-1401. CAMERON I. & SMITH R. (1964) Cytological responses of brown fat tissue in cold-exposed rats. ] . Cell. Biol. 23, 89-100. CHAFFEE R. R. J., ALL~'~ J., CASSUTOY. & SMITH R. (1964) Biochemistry of brown fat and liver in cold-acclimatized hamsters. Am. ]. Physiol. 207, 1211-1214. C ~ R. R. J., P~GELLEY E. T., ALLEN J. R. & SMITH R. E. (1966) Biochemistry of brown fat and liver of hibernating golden-mantled ground squirrels (Citellus lateralis). Can. ]. Physiol. Pharmac. 44, 217-223. DAWKINS M. J. R. & HULL D. (1964) Brown adipose tissue and the response of new-born rabbits to cold. ] . Physiol. 172, 216-238. FEIST D. D. & QUAY W. B. (1968) Norepinephrine content and sympathetic regulation in adipose and splenic tissues of hibernating and post-arousal hamsters. Fedn Proc. Fedn Am. Soc. exp. Biol. 27, 633. FOLCH J., LEES M. & SLO~-E-STANLEY G. H. (1957) Simple methods for isolation and purification of total lipids from animal tissues..~, biol. Chem. 226, 497-509. HAYWAm:~J. S. & LYMAN C. P. (1967) Nonshivering heat production during arousal from hibernation and evidence for the contribution of brown fat. In Mammalian Hibernation, Vol. I I I (Edited by FISHER K. C., DAW~ A. R., LYM~'~ C. P., SCHONBAUME. & SOUTH F. E., JR.), pp. 346-354. American Elsevier, New York. HIMMS-HAGEN J. (1965) Lipid metabolism in warm-acclimated and cold-acclimated rats exposed to cold. Can.]. Physiol. Pharmac. 43, 370--403. JOEL C. (1965) T h e physiological role of brown adipose tissue. In Handbook of Physiology, Section 5: ~tdipose Tissue (Edited by RSNOLD A. E. & CAmLL G. F. JR.), Chapter 9. American Physiological Society, Washington. JOEL C., TREBLE D. & BALL E. G. (1964) On a major role for brown adipose tissue in heat production during arousal from hibernation. Fedn Proc. Fedn Am Soc. exp. Biol. 23, 271. LowRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent..7, biol. Chem. 193, 265-275. LYMAN C. P. (1948) T h e oxygen consumption and temperature regulation of hibernating hamsters (Mesocricetus auratus). ]. exp. Zool. 109, 55-78. MASORO E. J. (1966) Effect of cold on metabolic use of lipids. Physiol. Rev. 46, 67-101. P R u s I ~ S. B., C A ~ O N B. & LINDBERC O. (1968) Oxidative metabolism in cells isolated from brown adipose tissue---I. Catecholamine and fatty acid stimulation of respiration. Eur.]. Biochem. 6, 15-22. ROBERTS J. C. & SMITH R. E. (1967) Time-dependent responses of brown fat in coldexposed rats. Am..~. Physiol. 212, 519-525. SIMPSON G. G., RoE A. & LEWONTIN R. C. (1960) Quantitative Zoology. Harcourt, Brace, New York. SMALLEY R. L. & DRYER R. L. (1967) Brown fat in hibernation. In Mammalian Hibernation, Vol. I I I (Edited by FISHER K. C., DAW~ A. R., LYMAN C. P., SCHONBAUME. & SOUTH F. E., JR.), pp. 325-345. American Elsevier, New York.
COLD ACCLIMATION AND HIBERNATION IN THE GOLDEN HAMSTER
119
SMIT G. J. & SMIT-VI$ J. H. (1966) Hibernation in the golden hamster, Mesocricetus auratus Waterhouse; A histological s t u d y - - I . Liver and interscapular brown adipose tissue. Arch. neerl. Zool. XVI 4, 453-468. SMITH R. E. & ROBERTS J. (1964) Thermogenesis of brown adipose tissue in cold acclimated rats. Am.J. Physiol. 206, 143-148. SOUMALAINEN P. • HERLEVI A. M. (1951) T h e alarm reaction and the hibernating gland. Science 114, 300. SPENCER W. A., GRODUMS E. I. & DEMPSTER G. (1966) T h e glyceride fatty acid composition and lipid content of brown and white adipose tissue of the hibernator Citellus lateralis. J. cell. Physiol. 67, 431 A,A.A. STEINER G. & CAHILL G. (1964) Brown and white adipose tissue metabolism in cold-exposed rats. Am.J. Physiol. 207, 840-844. THOMSON J. F., SMITH D. E., NANCE S. L. & HABECKD. A. (1968) Some metabolic characteristics of brown fat with particular reference to the mitochondria. Cornp. Biochem. Physiol. 25, 783-804. TREBLE D. H. & BALL E. G. (1963) T h e occurrence of glycerokinase in rat brown adipose tissue. Fedn Proc. Fedn Am. Soc. exp. Biol. 22, 357. WELLS H. J., MAKITA M., WELLS W. W. & KRUTZSCHP. H. (1965) A comparison of the lipid composition of brown adipose tissue from male and female bats (Myotis lucifugus) during hibernating and non-hibernating seasons. Biochim. biophys. Acta 98, 269-277.
Key Word Index--Arousal; cold acclimation; brown fat; hamsters; hibernation; lipids ; proteins; Mesocricetus auratus.
EFFECTS OF COLD ACCLIMATION AND AROUSAL FROM HIBERNATION ON BROWN FAT LIPID AND PROTEIN IN THE GOLDEN HAMSTER (MESOCRICETUS A URATUS) DALE D. F E I S T and W I L B U R B. QUAY Department of Zoology, University of California, Berkeley, California
(Received 21 February 1969) A b s t r a c t - - 1 . Total lipid and protein are monitored in interscapular, cervical
and thoracic brown fat deposits from normal control, cold-acclimated, hibernating and newly aroused golden hamsters (Mesocricetus auratus). 2. During cold acclimation total lipid declines significantly while protein content increases markedly in all brown fat deposits. In contrast to interscapular and cervical deposits, thoracic brown fat contains a higher percentage of protein than lipid after cold acclimation. 3. After cold acclimation brown fat retains the same lipid and protein levels in hamsters exposed to very short and very long hibernating periods. 4. Newly aroused hamsters show little or no evidence of loss of brown fat lipid during arousal from hibernation. This contrasts with findings of other workers that ground squirrels lose a substantial percentage of brown fat lipid during arousal. INTRODUCTION MUCH evidence now indicates that brown fat plays an important role in nonshivering thermogenesis during cold acclimation and arousal from hibernation in mammals (Joel, 1965; Smalley & Dryer, 1967). Of the various substrates which may be involved in the biochemical processes of thermogenesis lipids may be significant (Masoro, 1966; Prusiner et al., 1968). Investigators have reported changes in the amount and composition of lipids in brown fat in association with cold acclimation, hibernation, and arousal from hibernation. Both cytological (Cameron & Smith, 1964) and chemical (Steiner & Cahill, 1964) studies show a decrease in the amount of lipid during cold acclimation in rats. Higher lipid levels have been reported in bat brown fat during hibernation (Wells et al., 1965). Soumalainen & Herlevi (1951 ) found a decrease in size and number of sudanophilic particles in the hedgehog brown fat immediately after arousal from hibernation. A decrease in brown fat glyceride levels was observed in the ground squirrel (Citellus lateralis) after arousal from hibernation (Spencer et al., 1966). Joel et al. (1964) and Joel (1965) have reported a loss of about 50 per cent of the lipid in brown fat of the thirteenlined ground squirrel during arousal from hibernation. J o e l concluded that complete oxidation of the lipid lost during arousal could provide more than enough calories to warm the ground squirrel to complete arousal. 111
112
DALE D. FEIST AND WILBURB. QUAY
T h e work reported here was initiated in the course of investigations of norepinephrine metabolism in b r o w n fat of hibernating and non-hibernating hamsters (Feist & Quay, 1968). I t was felt to be of value to determine what changes, if ally, in total lipids and proteins in several different brown fat deposits accompany the processes of cold acclimation and arousal f r o m hibernation in the Syrian golden hamster. MATERIALS AND M E T H O D S Adult golden hamsters (Mesocricetus auratus) were housed in individual metal cages with wood-chip bedding in either a warm room at 22 + 1°C or a cold room at 5 + 1°C. They were maintained on 8 hr of artificial illumination per day and given Purina rat chow and water adlib. The animals were subdivided as follows: (1) normal control (NC) animals maintained at 22 + 1°C ; (2) cold-acclimated (C) hamsters kept at 5 + 1°C, having hibernated, but not in hibernation at the time of killing; (3) hibernating (H) animals in deep hibernation with a rectal and/or cheek pouch temperature of 5-7°C; (4) newly aroused (A) animals which have attained a rectal temperature of 36-38°C after artificial stimulation by taking of rectal or cheek pouch temperature to arouse from deep hibernation. Rectal and cheek pouch temperatures were monitored with a small animal thermistor probe attached to a Yellow Springs Instrument Company telethermometer. Experiments were conducted between the final week in March and the first week of September. The animals were killed between 10.30 a.m. and 3.15 p.m. by rapid decapitation. Interscapular brown fat, cervical brown fat (from both sides of the ventral midline in the mid-cervical region) and thoracic brown fat (from along the course of the thoracic segment of the posterior vena cava) were excised and frozen on dry ice. The tissues were stored in a dry-ice chest at - 4 0 ° C until analysis. The analysis of total lipids was performed essentially according to Folch et al. (1957). Protein content was assayed by the method of Lowry et al. (| 951). Wet weight of the tissues was determined on a torsion balance. Lipid and protein are expressed in terms of mg/g dry wt. of tissue on the assumption that combined lipid plus protein constitute the dry weight and that carbohydrates and nucleic acids make up a negligible percentage of the total dry weight (Stair & Smit-Vis, 1966; Thomson et al., 1968). The "Student" t-test was used to estimate the statistical significance of the difference between mean values (Simpson et al., 1960).
RESULTS Body weight changes during cold exposure T h e total b o d y weights of hamsters in three different experimental sets were maintained or increased in normal control animals and decreased in all animals exposed to the cold. T h e s e results can be seen in T a b l e 1 along with descriptive data for the three experimental sets. Each experiment contains normal controls (NC), cold-acclimated (C), hibernating (H) and newly aroused (A) animals (except experiment I I which only contains hibernating and aroused animals) and is based on the length of time elapsed f r o m the onset of the hibernation period to the time of autopsy and excision of tissues. T h e length of cold exposure is m u c h shorter in experiment I than in experiment I I I . T h e length of the hibernation period is shortest in experiment I and longest in experiment I I I . (Hibernation period as defined here represents a series of entries and arousals f r o m single bouts of deep hibernation.)
Males Males Females
I II III
5~ 10 11
16 8 24
N* 106+4(16) 109 + 2 (8) 102+3(24)
All
At start C
126+13(4) 99_+3(4) --1 0 0 + 6 ( 6 ) 89_+6(6)
NC t
89+3(4) 80 + 2 (4) 81+4(6)
H
At autopsy A 85+4(4) 82 + 2 (4) 78+4(6)
(Mean ~ standard error (N*))
* N = n u m b e r of animals. t Groups : NC = normal controls, C = cold-acclimated, H = hibernating, A = newly aroused.
Sex
Experiment No.
Age at autopsy (months)
Body weights (g)
TABLE 1 - - B O D Y WEIGHTS AND EXPERIMENTAL TREATMENTS
8-10 16 16
1-2 7-9 11-14
Cold Hibernation (re: C,H,A,) (re: H,A,)
Length (weeks) of
114
DALS D. FI~ISTAND WILBUR B. QUAY
Lipid and protein changes during cold acclimation Interscapular brown fat from normal control animals in experiment I (see Fig. 1) contains about 64 per cent lipid and 36 per cent protein/dry wt. of tissue. During cold acclimation there is a significant loss ( P < 0.05) of about 23 per cent of Intecscopulor Brown f'of;
PROTEIN
LIPID
_}*
70C
*P<.05
I
(NC vsC)
60C 50C
40C .~
30c 20C
IOC
I
-
I
o
Groups: NC
H A
NCC
!i H A
Fxo. 1. Lipid and protein content of interscapular brown fat in normal (NC), cold-acclimated (C), hibernating (H) and newly aroused (A) hamsters exposed to short cold acclimation and hibernating periods. Values given = mean + S.E. of the mean. [ ~ = LIPID
[]=PROTEIN
P: (NC vs C) ,x--x--,~< .001 "~"~ < ,01
90C
(H vs A] "x'< . 0 5
.~ ,11-,)t~ 80O
70C >" 600 "o O~ 500
.... I--Z'~
r~.-" 40O 300 .
!~.-~
200 IO0
0
Groups: NC C H A Tissue:
Inlerscopulor
NC C H A Brown Fat
NC C H A
NC C H A
Cervicol Brown For
NCCHA
NCCHA
Thorocic Brown Fo!
FIc. 2. Lipid and protein content of interscapular, cervical and thoracic brown fat in normal (NC), cold-acclimated (C), hibernating (H) and newly aroused (A) hamsters exposed to long cold acclimation and hibernating periods. Values given = mean + S.E. of the mean.
4
A
Change (%) P
N 4
Group H
TABLE 2--LIPID
- 3 N.S.
546.9 + 14.2
Lipid 565.9 + 12.9
+2 N.S.
453.1 + 14.2
Protein 434.1 + 12-9
Interscapular brown fat
- 3 N.S.
524.9 + 16.5
Lipid 540.3 + 13.0
+3 N.S.
475.1 + 16.5
(H)
AND N E W L Y AROUSED
+2 N.S.
409-2 + 29.5
Lipid 400.6 + 20-1
+ 1"5 N.S.
590.8 + 29.5
Protein 599.4 + 20-1
Thoracic brown fat
FROM H I B E R N A T I N G
Protein 459.7 + 13.0
Cervical brown fat
(mg/g DRY TISSUE) I N BROWN FAT DEPOSITS (A) HAMSTERS I N M I D - H I B E R N A T I O N PERIOD
AND P R O T E I N CONCENTRATIONS
tJi
116
DALE D. FEISTANDWILBURB. QUAY
the tissue lipid and a significant increase ( P < 0.05) in protein content of about 41 per cent. Interscapular brown fat and cervical brown fat from normal control hamsters in experiment III (see Fig. 2) contains about 80 per cent lipid to 20 per cent protein/dry wt., while thoracic brown fat contains 67 per cent lipid. During cold acclimation the amount of lipid diminishes significantly (see Fig. 2) by 30-40 per cent at all brown fat sites while protein increases markedly by 86--136 per cent. In the thoracic deposit the ratio of lipid to protein was almost completely reversed during cold acclimation. Thoracic brown fat from all cold-acclimated, hibernating and newly aroused animals always contained more protein than lipid.
Lipid and protein of brown fat in hibernating and aroused hamsters In hibernating hamsters from all three experiments (I, II, III), artificial stimulation by manual determination of rectal and cheek pouch temperatures induced arousal. Arousal from a body temperature of 5-6°C to 36-37°C at an ambient temperature of 5 + I°C was completed within 3-4 hr (usually between 3 and 3.5 hr) in all animals. In all three experiments (I, II, III), interscapular brown fat and cervical brown fat from newly aroused hamsters showed no significant change in the content of lipid or protein when compared with the same tissue from hibernators (see Figs. 1, 2; Table 2). Thoracic brown fat in group II (Table 2) showed no alteration in lipid content after arousal. But thoracic brown fat in group III (Fig. 2) lost 15.5 per cent of the lipid during arousal ( P < 0.05). DISCUSSION The decline in body weight which accompanies cold exposure in these hamsters is consistent with the findings of a number of investigators (Lyman, 1948; Chaffee et al., 1964; Smit & Smit-Vis, 1966). During this same period the absolute mass of brown fat has been shown to increase (unpublished results from this laboratory; Chaffee et aL, 1964). The concomitant increase in protein and fall in lipid content in the three brown fat deposits as shown here affirms the results previously reported in hamsters (Chaffee et al., 1964) and rats (Steiner & Cahill, 1964) during cold acclimation. Biochemical investigations of synthetic and oxidative metabolic activities of brown fat during cold acclimation in rats (Smith & Roberts, 1964; Steiner & Cahill, 1964; Himms-Hagen, 1965; Roberts & Smith, 1967), hamsters (Baumber & Denyes, 1964; Chaffee et al., 1964) and ground squirrels (Chaffee et al., 1966) have provided evidence for an enhanced thermogenic capacity. Presumably the changes in lipid and protein during cold acclimation are related to this enhancement. In this study there was no difference in the percentage of lipid in brown fat deposits of cold-acclimated and hibernating hamsters killed early in the hibernating period from those killed late in the hibernating period. Smit & Smit-Vis (1966) observed histochemically only a slight decrease in lipid of interscapular brown fat in the later stages of the hibernating period of the hamster. They suggest that it is not likely that brown adipose tissue has much nutritive value during lethargy and arousal.
COLD A C C L I M A T I O N AND H I B E R N A T I O N I N T H E G O L D E N HAMSTER
117
The demonstration here that the percentage of total lipid does not decline significantly in any brown fat deposit at the end of arousal (except for a 15.5 per cent loss of lipid in thoracic brown fat in experiment III) differs from results reported in two species of ground squirrels. Spencer et al. (1966) report losses of between 25 and 38 per cent of brown fat glycerides at the end of arousal in Citellus lateralis. Joel (1965) describes a loss of about 50 per cent of the brown fat lipid in newly aroused Citellus tridecemlineatus. The lipid level was shown to return to near normal by 15--46 hr after completion of arousal. Joel also found an increase in plasma free fatty acids and glycerol during arousal. He has suggested that brown fat may perform two quantitatively important roles during arousal from hibernation: (1) oxidation of lipid to produce heat within the tissue; (2) release of large amounts of substrates into the circulation for utilization by other tissues. If brown fat in arousing hamsters performs these roles, one might expect to find a concomitant marked decrease in lipid as found in the ground squirrel. It is possible that there is a rapid loss of lipid during arousal combined with an equally rapid replacement which would resuk in no apparent loss. Another possibility is that during arousal in hamsters, thermogenesis in brown fat may result (at least in part) from internal hydrolysis and resynthesis of triglycerides with little or no loss of free fatty acids and glycerol. This would seem to be supported by evidence for accelerated triglyceride cycling--which may act as an ATPase system for heat production--in brown fat of cold-acclimated rats (Ball & Jungus, 1961; HimmsHagen, 1965) and new-born rabbits (Dawkins & Hull, 1964). Glycerol kinase, essential for re-esterification of glycerol and maintenance of triglyceride recycling, has been demonstrated in brown fat from rats and ground squirrels (Treble & Ball, 1963; Joel, 1965). It may be operative in hamster brown fat. It also seems possible that the quantitative contribution of brown fat (or brown fat lipid) to the total thermogenic response relative to the contribution of other nonshivering and shivering tissues may not be as great in the arousing hamster as in other species of hibernators. Attempts at quantitative estimation of the calorie contribution of brown fat to the total warming process based on in vitro and in vivo experiments have not yet clarified this matter (see Hayward & Lyman, 1967; Smalley & Dryer, 1967; Thomson et al., 1968). Hayward & Lyman (1967) have reported from studies of curarized and non-curarized animals that while brown fat contributes to warming, shivering is essential to normal arousal in hamsters and dormice but not in bats. They do not give any information on the importance of shivering and brown fat in ground squirrels. It would be of interest to know how much lipid is lost from bat brown fat during arousal. These possibilities remain to be tested in the arousing hamster in order to elucidate the role of brown fat and its lipid. Acknowledgements--The authors wish to express their appreciation to Dr. Glenn D. Bissel, State Health Department, Fairfield, California, for generously providing the hamsters used in this study, to Mr. William Young for animal room assistance and to Mrs. Emily Reid for final rendering of the figures.
118
DALE D. FEIST AND WILBtm B. QUAY
This work was supported in part by U.S.P.H.S. predoctoral fellowship G M 5-Fl-GM-32,590 (to D. D. F.) and research funds, University of California Berkeley (to W. B. Q.). REFERENCES BALL E. G. & JUNGUS R. (1961) On the action of hormones which accelerate the rate of oxygen consumption and fatty acid release in rat adipose tissue in vitro. Proc. hath. Acad. ScL U.S.A. 47, 932-941. BAUMBERJ. & DENYES A. (1964) A c e t a t e - l - C utilization by brown fat from hamsters in cold exposure and hibneration. Can.]. Biochem. 42, 1397-1401. CAMERON I. & SMITH R. (1964) Cytological responses of brown fat tissue in cold-exposed rats. ] . Cell. Biol. 23, 89-100. CHAFFEE R. R. J., ALL~'~ J., CASSUTOY. & SMITH R. (1964) Biochemistry of brown fat and liver in cold-acclimatized hamsters. Am. ]. Physiol. 207, 1211-1214. C ~ R. R. J., P~GELLEY E. T., ALLEN J. R. & SMITH R. E. (1966) Biochemistry of brown fat and liver of hibernating golden-mantled ground squirrels (Citellus lateralis). Can. ]. Physiol. Pharmac. 44, 217-223. DAWKINS M. J. R. & HULL D. (1964) Brown adipose tissue and the response of new-born rabbits to cold. ] . Physiol. 172, 216-238. FEIST D. D. & QUAY W. B. (1968) Norepinephrine content and sympathetic regulation in adipose and splenic tissues of hibernating and post-arousal hamsters. Fedn Proc. Fedn Am. Soc. exp. Biol. 27, 633. FOLCH J., LEES M. & SLO~-E-STANLEY G. H. (1957) Simple methods for isolation and purification of total lipids from animal tissues..~, biol. Chem. 226, 497-509. HAYWAm:~J. S. & LYMAN C. P. (1967) Nonshivering heat production during arousal from hibernation and evidence for the contribution of brown fat. In Mammalian Hibernation, Vol. I I I (Edited by FISHER K. C., DAW~ A. R., LYM~'~ C. P., SCHONBAUME. & SOUTH F. E., JR.), pp. 346-354. American Elsevier, New York. HIMMS-HAGEN J. (1965) Lipid metabolism in warm-acclimated and cold-acclimated rats exposed to cold. Can.]. Physiol. Pharmac. 43, 370--403. JOEL C. (1965) T h e physiological role of brown adipose tissue. In Handbook of Physiology, Section 5: ~tdipose Tissue (Edited by RSNOLD A. E. & CAmLL G. F. JR.), Chapter 9. American Physiological Society, Washington. JOEL C., TREBLE D. & BALL E. G. (1964) On a major role for brown adipose tissue in heat production during arousal from hibernation. Fedn Proc. Fedn Am Soc. exp. Biol. 23, 271. LowRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent..7, biol. Chem. 193, 265-275. LYMAN C. P. (1948) T h e oxygen consumption and temperature regulation of hibernating hamsters (Mesocricetus auratus). ]. exp. Zool. 109, 55-78. MASORO E. J. (1966) Effect of cold on metabolic use of lipids. Physiol. Rev. 46, 67-101. P R u s I ~ S. B., C A ~ O N B. & LINDBERC O. (1968) Oxidative metabolism in cells isolated from brown adipose tissue---I. Catecholamine and fatty acid stimulation of respiration. Eur.]. Biochem. 6, 15-22. ROBERTS J. C. & SMITH R. E. (1967) Time-dependent responses of brown fat in coldexposed rats. Am..~. Physiol. 212, 519-525. SIMPSON G. G., RoE A. & LEWONTIN R. C. (1960) Quantitative Zoology. Harcourt, Brace, New York. SMALLEY R. L. & DRYER R. L. (1967) Brown fat in hibernation. In Mammalian Hibernation, Vol. I I I (Edited by FISHER K. C., DAW~ A. R., LYMAN C. P., SCHONBAUME. & SOUTH F. E., JR.), pp. 325-345. American Elsevier, New York.
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SMIT G. J. & SMIT-VI$ J. H. (1966) Hibernation in the golden hamster, Mesocricetus auratus Waterhouse; A histological s t u d y - - I . Liver and interscapular brown adipose tissue. Arch. neerl. Zool. XVI 4, 453-468. SMITH R. E. & ROBERTS J. (1964) Thermogenesis of brown adipose tissue in cold acclimated rats. Am.J. Physiol. 206, 143-148. SOUMALAINEN P. • HERLEVI A. M. (1951) T h e alarm reaction and the hibernating gland. Science 114, 300. SPENCER W. A., GRODUMS E. I. & DEMPSTER G. (1966) T h e glyceride fatty acid composition and lipid content of brown and white adipose tissue of the hibernator Citellus lateralis. J. cell. Physiol. 67, 431 A,A.A. STEINER G. & CAHILL G. (1964) Brown and white adipose tissue metabolism in cold-exposed rats. Am.J. Physiol. 207, 840-844. THOMSON J. F., SMITH D. E., NANCE S. L. & HABECKD. A. (1968) Some metabolic characteristics of brown fat with particular reference to the mitochondria. Cornp. Biochem. Physiol. 25, 783-804. TREBLE D. H. & BALL E. G. (1963) T h e occurrence of glycerokinase in rat brown adipose tissue. Fedn Proc. Fedn Am. Soc. exp. Biol. 22, 357. WELLS H. J., MAKITA M., WELLS W. W. & KRUTZSCHP. H. (1965) A comparison of the lipid composition of brown adipose tissue from male and female bats (Myotis lucifugus) during hibernating and non-hibernating seasons. Biochim. biophys. Acta 98, 269-277.
Key Word Index--Arousal; cold acclimation; brown fat; hamsters; hibernation; lipids ; proteins; Mesocricetus auratus.