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Cold acclimation in food-restricted rats
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Camp. Biochem. Physiol. Vol. 87A, No. I, pp. 31-33, 1987 Printedin Great Britain
COLD ACCLIMATION
C 1987Pergamon Journals
IN FOOD-RESTRICTED
RATS
M. L. PUERTA and M. ABELEKDA Departamento de Fisiologia Animal, Fact&ad de Ciencias Biologicas, Universidad Complutense de Madrid, 28040, Spain (Received
2 July 1986)
Abstract-l. Food intake, body weight and brown adipose tissue (BAT) mass and composition of rats exposed at 6’C either with food ud libifum or food-restricted were compared with those of rats in the thermoneutral zone, with food ad fibitum. 2. Cold acclimation with food ad libifum increases food intake and prevents body weight gains. IBAT (interscapular BAT) increases its mass and changes its composition after 3 weeks of cold exposure. 3. Cold acclimation with food restriction produces a progressive decrease in body weight. IBAT mass increases after 3 weeks but changes in composition occur sooner. 4. It is concluded that the overfeeding that accompanies cold acclimation is not necessary for non-shivering thermogenesis in BAT.
INTRODUCTION
Comparison f-test.
In 1979, Foster and Frydman demonstrated that brown adipose tissue (BAT) is the main site of cold-induced non-shivering thermogenesis (CIT). Later, Rothwell and Stock (1980) demonstrated that BAT is also responsible for adaptative diet-induced thermogenesis (DIT). Thus cold and hyperphagia are the physiological signals that induce the adaptative changes in BAT, nominally, hypertrophy, hyperplasia, increase in proteinic and decrease in lipidic content. Besides, during the thermogenic condition, oxidative phosphorylation is uncoupled, allowing the oxidation of substrates to proceed without ATP synthesis. In 1977, Kuroshima et (11. observed an increased food intake in cold acclimated tats and Rothwell and Stock (1980) obtained similar results. Since voluntary overfeeding produces adaptative changes in BAT and cold acclimated rats are hyperphagic, it can be argued that cold induces BAT changes by previously inducing overfeeding. Our work was undertaken to study this point. MATERIALS AND
between
groups
was done
by a Student’s
RESULTS
Figure I depicts daily food intake of both groups of animals fed ad /i&rum. At thermoneutrality (28°C) rats eat 18 g daily. Cold exposure is followed by a severe reduction in food intake which takes place between 2448 hr after cold entry. Several days later, rats increase their food intake. Therefore, although cold enhances food intake it takes about 1 week to reach a hyperphagic state. The alterations from the initial body weight are represented in Fig. 2. Warm-exposed animals, which show a regular daily food intake, also increase their body weight linearly. However, cold exposure with food ad fibitum is followed by an initial decrease in body weight that parallels the initial reduction in food intake. This weight loss stops when hyperphagia begins. Cold-exposed food-restricted rats also lose weight but two different phases can be distinguished. There is a rapid fall when cold exposure begins (and food intake is almost suppressed) followed by a discrete but continuous loss probably due to food restriction. On the other hand, cold exposure modifies IBAT mass and composition in both groups of cold-exposed rats but at different rates (Table I). Hypertrophy is accomplished on the third week in both groups but changes in IBAT composition occur sooner in coldexposed, food-restricted rats.
METHODS
Male Sprague-Dawley rats (Rorrusnort;egicu.s), 226233 g of body weight at the beginning of the experiment, were subjected to one of the following conditions: (a) 28’C and food ad libitum (warm-exposed rats, WE); (b) 6°C and food ad libifum (cold-exposed rats, CE); (c) 6’C and daily food restriction (80% of WE intake: cold-exposed, foodrestricted rats. CEFR). Food was a commercial laboratory stock diet (Panlab). All animals were in individual cages with free access to water and with a light schedule 12: 12. Food intake and body weight was monitored daily. Half of the animals were sacrificed by decapitation 2 or 3 weeks respectively after the beginning of the experiment. Interscapular brown adipose tissue (IBAT) was excised and placed in ice saline. After removal of the adhering white adipose tissue and muscle, IBAT was weighed and stored at -20°C until analysis. Total lipids were extracted from one of the IBAT pads according to Folch el 01. (1957) and were weighed after evaporating to vacuum the chloroformic phase. Total proteins and nucleic acids were assayed in the other pad after the method of Lowry ef ul. (1951) and Schneider’s procedure (1957). respectively.
DISCUSSION
If cold induces BAT adaptative changes directly or inducing hyperphagia previously was questioned after the observation of hyperphagia in cold acclimated rats. Johnson et al. (1982) observed that coldexposed rats pair fed with warm controls (24°C) did not increase IBAT mass despite the fact that this tissue was receiving a similar sympathetic activation than that received by IBAT of cold exposed rats fed 31
32
M. L. ~ERTA
and M. ABELENDA
WE
1
3
Fig. 1. Daily food intake respectively). Each datum
Fig. 2. Changes CE, respectively)
5
7
9
11
13
15
17
19
”
OAYS
of warm- (28T) and cold- (6’C) exposed rats fed ad libirum (WE and CE, is M f SE of five to ten animals. P < 0.001 on day 7 and the following days.
in body weight in warm- (28°C) and cold- (6,‘C) exposed rats fed ad libifum (WE and and in cold-exposed, food-restricted rats (CEFR). Each datum is M & SE of five to ten animals.
ad libitum. They suggested a limitation
of substrates as the limiting factor for BAT hypertrophy. However, Kuroshima and Yahata (1985) did find the same IBAT mass and gross composition in cold exposed rats either fed ad libitum or pair fed with
warm controls (25°C). Besides, both groups of animals showed the same cold tolerance at - 5°C. They concluded that cold itself is enough to produce non-shivering thermogenesis indepcndcntly of food intake. There are several differences between the
Table I. IBAT mass and composition of warm-exposed rats (28’C) fedad libirum
(WE), cold-exposed raps (6'C)fedad lihirum (CE) and cold-exposed rats (UC), food-restricted (CEFR) after 14 or 21 days of exposure
DAY 14
ng IBAT g IBAT/lOOg b.w.
DAY 21
KE
CE
CEFR
YE
240.00+16.27
279.88L19.30
263.74~6.84
250.8Ok19.75
CE 364.08i9.54'
CEFR 364.92215.76
C.103iO.006
C.126+0.008'
0.138+0.004c
lipids
47.74i2.51
29.77L2.43'
20.05?_1.67
proteins
10.14iO.56
9.9620.63
14.90+o.53c
10.6320.63
11.55+o.43a
12.81~0.25~
DiJA
c).176+0.010
C.178+0.013
0.220+0.006b
0.1c0+0.007
0.140t0.011a
0.186~0.011
RNA
0.210+0.015
C.270~0.025
o.392+o.014c
0.204+0.010
0.438+0.050b
0.320+0.009
0.100i0.005
0.168+0.00Zc
0.188f0.005
48.2322.77
28.67~2.50'
22.53+1.00'
mg/lOO 3g IBAT
Each datum isM I SE of live animals. ‘P < 0.05;bP
< 0.01;
c
‘P
< 0.001
when compared with warm cootrok
33
Cold acclimation in rats
cited. Johnson et al. (1982) used female Sprague-Dawley rats exposed to cold over 2 weeks, while Kuroshima and Yahata (1985) used male Wistar rats exposed to cold for 5 weeks. Since Puerta et al. (1984) have not detected sexual differences in cold acclimation of Sprague-Dawley rats two reasons can be the cause of the differences observed: the different strains used, or the different duration of both experiments. On analyzing the problem on a temporal basis it can be seen that cold-exposed Sprague-Dawley rats fed ad libitum become hyperphagic but the adaptative changes in BAT only take place after 3 weeks of cold exposure, i.e. when hyperphagia and cold have been acting together for at least 2 weeks. However, when food is restricted in the cold, hypertrophy also appears after 3 weeks of cold exposure, but changes in composition are accelerated and appear before hypertrophy occurs.
SUMMARY
works
Therefore
IBAT mass and composition
are similar
in cold-exposed male Sprague-Dawley rats either fed ad libitum or food-restricted, after 3 weeks of cold exposure. These results are in agreement with those obtained by Kuroshima and Yahata (1985) in male Wistar rats after 5 weeks of cold exposure. However, adaptative changes in IBAT composition are accelerated by food restrictions as observed by Johnson et nl. (1982) in female Sprague-Dawley rats, who dcscribed an increase in IBAT proteinic content. Since sympathetic stimulation and cold tolerance are similar in both groups of cold-exposed rats (Johnson et al., 1982; Kuroshima and Yahata, 1985), we believe that all the observations made tend to support the fact that cold itself determines adaptative changes in BAT although food restriction accelerates some of them [perhaps because of the increase in blood cetonic bodies and their utilization by BAT (Wright and Agius, 1983)].
In conclusion, cold acclimation is accompanied by an increase in food intake but such overfeeding is not necessary for acclimation to occur, as judged by the adaptative changes in BAT of cold-acclimated, foodrestricted rats. REFERENCES
Folch J., Less M. and Sloane-Stanley G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. hiol. Chem. 226, 497-509. Foster D. 0. and Frydman M. L. (1979) Tissue distribution of cold-induced thermogenesis in conscious warm- or cold-acclimated rats re-evaluated from changes in tissue blood Row: the dominant role of brown adipose tissue in the replacement of shivering by non-shivering thermogenesis. Can J. Physiol. Pharmac. 51, 257-270. Johnson T. S., Murray S., Young J. B. and Landsberg L. (1982) Restricted food intake limits brown adipose tissue hypertrophy in cold exposure. Life Sri. 30, 1423-1426. Kuroshima A., Katsuhiko D. and Yahata T. (1977) Improved cold tolerance and its mechanism in coldacclimated rats by high fat diet feeding. Can J. Physiol. Pharmac. 55, 943-950.
Kuroshima A. and Yahata T. (1985) Effect of food restriction on cold adaptability of rats. Can. J. Physiol. Pharmac. 63: 68-7
I,
Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Puerta M. L., Abelenda M. and Fraile A. (1984) Effects of thyroxine and 3,5,3’-triiodothyronine in brown adipose tissue of rats. Comp. Biochem: Physiol. 19A, 563-586. Rothwell N. J. and Stock M. J. (1980) Similarities between cold- and diet-induced thermogenesis in the rat. Can. J. Physiol. Pharmac. 58, 842-848:
Schneider W. C. (1957) Determination of nucleic acids in tissues by pent&e analysis. h4eth. Enzym. 3, 68CM84. Wright J. and Agius L. (1983) Fatty acid synthesis and ketone body utilization by brown adipose tissue of the rat. Biochim. biophys. Acta 753, 244-248.
Camp. Biochem. Physiol. Vol. 87A, No. I, pp. 31-33, 1987 Printedin Great Britain
COLD ACCLIMATION
C 1987Pergamon Journals
IN FOOD-RESTRICTED
RATS
M. L. PUERTA and M. ABELEKDA Departamento de Fisiologia Animal, Fact&ad de Ciencias Biologicas, Universidad Complutense de Madrid, 28040, Spain (Received
2 July 1986)
Abstract-l. Food intake, body weight and brown adipose tissue (BAT) mass and composition of rats exposed at 6’C either with food ud libifum or food-restricted were compared with those of rats in the thermoneutral zone, with food ad fibitum. 2. Cold acclimation with food ad libifum increases food intake and prevents body weight gains. IBAT (interscapular BAT) increases its mass and changes its composition after 3 weeks of cold exposure. 3. Cold acclimation with food restriction produces a progressive decrease in body weight. IBAT mass increases after 3 weeks but changes in composition occur sooner. 4. It is concluded that the overfeeding that accompanies cold acclimation is not necessary for non-shivering thermogenesis in BAT.
INTRODUCTION
Comparison f-test.
In 1979, Foster and Frydman demonstrated that brown adipose tissue (BAT) is the main site of cold-induced non-shivering thermogenesis (CIT). Later, Rothwell and Stock (1980) demonstrated that BAT is also responsible for adaptative diet-induced thermogenesis (DIT). Thus cold and hyperphagia are the physiological signals that induce the adaptative changes in BAT, nominally, hypertrophy, hyperplasia, increase in proteinic and decrease in lipidic content. Besides, during the thermogenic condition, oxidative phosphorylation is uncoupled, allowing the oxidation of substrates to proceed without ATP synthesis. In 1977, Kuroshima et (11. observed an increased food intake in cold acclimated tats and Rothwell and Stock (1980) obtained similar results. Since voluntary overfeeding produces adaptative changes in BAT and cold acclimated rats are hyperphagic, it can be argued that cold induces BAT changes by previously inducing overfeeding. Our work was undertaken to study this point. MATERIALS AND
between
groups
was done
by a Student’s
RESULTS
Figure I depicts daily food intake of both groups of animals fed ad /i&rum. At thermoneutrality (28°C) rats eat 18 g daily. Cold exposure is followed by a severe reduction in food intake which takes place between 2448 hr after cold entry. Several days later, rats increase their food intake. Therefore, although cold enhances food intake it takes about 1 week to reach a hyperphagic state. The alterations from the initial body weight are represented in Fig. 2. Warm-exposed animals, which show a regular daily food intake, also increase their body weight linearly. However, cold exposure with food ad fibitum is followed by an initial decrease in body weight that parallels the initial reduction in food intake. This weight loss stops when hyperphagia begins. Cold-exposed food-restricted rats also lose weight but two different phases can be distinguished. There is a rapid fall when cold exposure begins (and food intake is almost suppressed) followed by a discrete but continuous loss probably due to food restriction. On the other hand, cold exposure modifies IBAT mass and composition in both groups of cold-exposed rats but at different rates (Table I). Hypertrophy is accomplished on the third week in both groups but changes in IBAT composition occur sooner in coldexposed, food-restricted rats.
METHODS
Male Sprague-Dawley rats (Rorrusnort;egicu.s), 226233 g of body weight at the beginning of the experiment, were subjected to one of the following conditions: (a) 28’C and food ad libitum (warm-exposed rats, WE); (b) 6°C and food ad libifum (cold-exposed rats, CE); (c) 6’C and daily food restriction (80% of WE intake: cold-exposed, foodrestricted rats. CEFR). Food was a commercial laboratory stock diet (Panlab). All animals were in individual cages with free access to water and with a light schedule 12: 12. Food intake and body weight was monitored daily. Half of the animals were sacrificed by decapitation 2 or 3 weeks respectively after the beginning of the experiment. Interscapular brown adipose tissue (IBAT) was excised and placed in ice saline. After removal of the adhering white adipose tissue and muscle, IBAT was weighed and stored at -20°C until analysis. Total lipids were extracted from one of the IBAT pads according to Folch el 01. (1957) and were weighed after evaporating to vacuum the chloroformic phase. Total proteins and nucleic acids were assayed in the other pad after the method of Lowry ef ul. (1951) and Schneider’s procedure (1957). respectively.
DISCUSSION
If cold induces BAT adaptative changes directly or inducing hyperphagia previously was questioned after the observation of hyperphagia in cold acclimated rats. Johnson et al. (1982) observed that coldexposed rats pair fed with warm controls (24°C) did not increase IBAT mass despite the fact that this tissue was receiving a similar sympathetic activation than that received by IBAT of cold exposed rats fed 31
32
M. L. ~ERTA
and M. ABELENDA
WE
1
3
Fig. 1. Daily food intake respectively). Each datum
Fig. 2. Changes CE, respectively)
5
7
9
11
13
15
17
19
”
OAYS
of warm- (28T) and cold- (6’C) exposed rats fed ad libirum (WE and CE, is M f SE of five to ten animals. P < 0.001 on day 7 and the following days.
in body weight in warm- (28°C) and cold- (6,‘C) exposed rats fed ad libifum (WE and and in cold-exposed, food-restricted rats (CEFR). Each datum is M & SE of five to ten animals.
ad libitum. They suggested a limitation
of substrates as the limiting factor for BAT hypertrophy. However, Kuroshima and Yahata (1985) did find the same IBAT mass and gross composition in cold exposed rats either fed ad libitum or pair fed with
warm controls (25°C). Besides, both groups of animals showed the same cold tolerance at - 5°C. They concluded that cold itself is enough to produce non-shivering thermogenesis indepcndcntly of food intake. There are several differences between the
Table I. IBAT mass and composition of warm-exposed rats (28’C) fedad libirum
(WE), cold-exposed raps (6'C)fedad lihirum (CE) and cold-exposed rats (UC), food-restricted (CEFR) after 14 or 21 days of exposure
DAY 14
ng IBAT g IBAT/lOOg b.w.
DAY 21
KE
CE
CEFR
YE
240.00+16.27
279.88L19.30
263.74~6.84
250.8Ok19.75
CE 364.08i9.54'
CEFR 364.92215.76
C.103iO.006
C.126+0.008'
0.138+0.004c
lipids
47.74i2.51
29.77L2.43'
20.05?_1.67
proteins
10.14iO.56
9.9620.63
14.90+o.53c
10.6320.63
11.55+o.43a
12.81~0.25~
DiJA
c).176+0.010
C.178+0.013
0.220+0.006b
0.1c0+0.007
0.140t0.011a
0.186~0.011
RNA
0.210+0.015
C.270~0.025
o.392+o.014c
0.204+0.010
0.438+0.050b
0.320+0.009
0.100i0.005
0.168+0.00Zc
0.188f0.005
48.2322.77
28.67~2.50'
22.53+1.00'
mg/lOO 3g IBAT
Each datum isM I SE of live animals. ‘P < 0.05;bP
< 0.01;
c
‘P
< 0.001
when compared with warm cootrok
33
Cold acclimation in rats
cited. Johnson et al. (1982) used female Sprague-Dawley rats exposed to cold over 2 weeks, while Kuroshima and Yahata (1985) used male Wistar rats exposed to cold for 5 weeks. Since Puerta et al. (1984) have not detected sexual differences in cold acclimation of Sprague-Dawley rats two reasons can be the cause of the differences observed: the different strains used, or the different duration of both experiments. On analyzing the problem on a temporal basis it can be seen that cold-exposed Sprague-Dawley rats fed ad libitum become hyperphagic but the adaptative changes in BAT only take place after 3 weeks of cold exposure, i.e. when hyperphagia and cold have been acting together for at least 2 weeks. However, when food is restricted in the cold, hypertrophy also appears after 3 weeks of cold exposure, but changes in composition are accelerated and appear before hypertrophy occurs.
SUMMARY
works
Therefore
IBAT mass and composition
are similar
in cold-exposed male Sprague-Dawley rats either fed ad libitum or food-restricted, after 3 weeks of cold exposure. These results are in agreement with those obtained by Kuroshima and Yahata (1985) in male Wistar rats after 5 weeks of cold exposure. However, adaptative changes in IBAT composition are accelerated by food restrictions as observed by Johnson et nl. (1982) in female Sprague-Dawley rats, who dcscribed an increase in IBAT proteinic content. Since sympathetic stimulation and cold tolerance are similar in both groups of cold-exposed rats (Johnson et al., 1982; Kuroshima and Yahata, 1985), we believe that all the observations made tend to support the fact that cold itself determines adaptative changes in BAT although food restriction accelerates some of them [perhaps because of the increase in blood cetonic bodies and their utilization by BAT (Wright and Agius, 1983)].
In conclusion, cold acclimation is accompanied by an increase in food intake but such overfeeding is not necessary for acclimation to occur, as judged by the adaptative changes in BAT of cold-acclimated, foodrestricted rats. REFERENCES
Folch J., Less M. and Sloane-Stanley G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. hiol. Chem. 226, 497-509. Foster D. 0. and Frydman M. L. (1979) Tissue distribution of cold-induced thermogenesis in conscious warm- or cold-acclimated rats re-evaluated from changes in tissue blood Row: the dominant role of brown adipose tissue in the replacement of shivering by non-shivering thermogenesis. Can J. Physiol. Pharmac. 51, 257-270. Johnson T. S., Murray S., Young J. B. and Landsberg L. (1982) Restricted food intake limits brown adipose tissue hypertrophy in cold exposure. Life Sri. 30, 1423-1426. Kuroshima A., Katsuhiko D. and Yahata T. (1977) Improved cold tolerance and its mechanism in coldacclimated rats by high fat diet feeding. Can J. Physiol. Pharmac. 55, 943-950.
Kuroshima A. and Yahata T. (1985) Effect of food restriction on cold adaptability of rats. Can. J. Physiol. Pharmac. 63: 68-7
I,
Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Puerta M. L., Abelenda M. and Fraile A. (1984) Effects of thyroxine and 3,5,3’-triiodothyronine in brown adipose tissue of rats. Comp. Biochem: Physiol. 19A, 563-586. Rothwell N. J. and Stock M. J. (1980) Similarities between cold- and diet-induced thermogenesis in the rat. Can. J. Physiol. Pharmac. 58, 842-848:
Schneider W. C. (1957) Determination of nucleic acids in tissues by pent&e analysis. h4eth. Enzym. 3, 68CM84. Wright J. and Agius L. (1983) Fatty acid synthesis and ketone body utilization by brown adipose tissue of the rat. Biochim. biophys. Acta 753, 244-248.