- Email: [email protected]
Sequential changes in brown adipose tissue composition, cytochrome oxidase activity and GDP binding throughout pregnancy and lactation in the rat
Biochimica et Biophysica Acta
882 (1986) 187-191
187
Elsevier BBA22341
S e q u e n t i a l c h a n g e s in b r o w n a d i p o s e t i s s u e c o m p o s i t i o n , c y t o c h r o m e o x i d a s e a c t i v i t y a n d G D P b i n d i n g t h r o u g h o u t p r e g n a n c y a n d l a c t a t i o n in t h e r a t Francesc Villarroya, Antonio Felipe and Teresa Mampel
*
C,~tedra de Fisiologia General de la Facultat de Biologia, Unioersitat de Barcelona, Avinguda Diagonal 645, 08071 Barcelona (Spain)
(ReceivedJanuary20th, 1986)
Key words: Cytochromeoxidase; GDP binding; Lipid composition; Pregnancy; Lactation; (Rat brown adipose) The sequential appearance of changes in interscapular brown adipose tissue composition, cytochrome oxidase activity and GDP binding was studied throughout pregnancy and lactation in the rat. Brown adipose tissue was hypertrophied during pregnancy because of progressive lipid accumulation, whereas its mitochondrial component and G D P binding to brown fat mitochondria were unchanged. In early lactation (day S) there was a decrease in the overall GDP binding to brown fat only because of the lower mitochondrial protein content. In late stages of lactation (days 10 and IS), the amount of tissue and its mitochondrial protein content were minimal and the G D P binding per mitochondrial protein decreased substantially. Scatchard analysis in day-IS-lactating rats indicated a large decrease in G D P binding sites without any changes in affinity. It is concluded that the diminished thermogenic activity of brown fat in lactation is attained through changes at different structural levels of the tissue occurring in a characteristic sequential trend; first a reduction in its mitochondrial component, and only later, at mid-lactation, a decrease in the specific mitochondrial proton conductance pathway activity.
Introduction
Brown adipose tissue has been recognized as the main site for both diet [1] and cold-induced [2] non-shivering thermogenesis in rodents. Thermogenesis in brown fat is due to the proton conductance pathway [3] that allows protons to translocate through the inner mitochondrial membrane without coupling to ATP synthesis. The activity of this pathway can be measured by determining the specific binding of G D P to brown adipose tissue mitochondria, due to the property of purine nucleotides to bind to a M r 32 000 mitochondrial protein inhibiting the proton conductance pathway. The high thermogenic activity of brown adipose * To whom correspondenceshould be addressed.
tissue in the cold-acclimated or chronically overfed animals is achieved through increases in the activity of the mitochondrial proton conductance pathway together with an increment in the mitochondria content and a general hypertrophy a n d / o r hyperplasia of the tissue [4,5]. On the other hand, brown fat thermogenesis has been reported to be impaired in animals with lowered energy expenditure and subsequently obese, such as the o b / o b and d b / d b mice and the f a / f a rat. The defective brown adipose thermogenic activity in these animals is associated with a diminished mitochondrial G D P binding [6-8], and a decrease in the mitochondrial component of the tissue has also been reported in the o b / o b [8] and d b / d b mice [6]. Much less is known about functional modifications in the brown adipose tissue under physio-
0304-4165/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
188 logical situations associated with substantial changes in energy intake and energy expenditure such as those of the breeding cycle. Trayhurn et al. [9] have described diminished activity of the proton conductance pathway in brown fat from pregnant and lactating mice, together with a decrease in the oxidative capacity of the tissue assessed by the cytochrome oxidase activity. However, studies on lipogenesis, considered by some authors to be closely linked to thermogenesis in brown fat [10] have shown greater lipogenesis and increased fatfree mass in the tissue from pregnant rats, as well as diminished lipogenesis without signs of hypotrophy in the mid-lactating rat [11]. The aim of the present work is to study further the changes in brown adipose tissue composition and thermogenic activity during pregnancy and lactation by means of determining the appearance in time of brown fat changes throughout the different stages of the breeding cycle in the rat. Materials and Methods
Female Wistar rats with an initial weight of 210-220 g were used. They were mated with adult males and the day of pregnancy was determined by the presence of spermatozoa in vaginal smears. After fecundation the rats were kept in individual cages, and when lactating rats were studied litter sizes were adjusted to ten pups. The animals were maintained under automatically controlled temperature conditions (23 + 1 °C) and 12-h light-dark cycles. They were fed ad libitum on a stock diet (A04 Panlab, Barcelona, Spain). All experiments were started between 09:00 and 10:00 h. Pregnant rats of 15, 18 and 20 days of gestation, lactating rats 5, 10 and 15 days after parturition and virgin controls were killed by decapitation. The interscapular brown adipose tissue was rapidly removed and dissected free of any adhering connective tissue or white adipose tissue. It was weighed and a small sample was used for lipid determination carried out as described by Folch et al. [12]. The remaining tissue was homogenized in 0.25 M sucrose, and mitochondria were prepared as described by Cannon and Lindberg [13]. The purine nucleotide binding assay was performed at room temperature (22°C) using [8-3H]GDP (Amersham, U.K.) using the method of Nicholls
[3] as modified by Goodbody and Trayhurn [6]. Scatchard analysis of [3H]GDP binding was performed as described above with concentrations ranging from 0.6 to 20 ~M. Nonspecific binding, determined by the presence of 100 #M GDP, was negligible. The protein content of mitochondrial preparations and the original tissue homogenate was determined [14] and the cytochrome oxidase activity (EC 1.9.3.1) was measured by a polarographic method [15]. The recovery of cytochrome oxidase activity in the mitochondrial preparations was calculated in each sample and its value was used to estimate the amount of mitochondrial protein in the interscapular brown adipose tissue. Student's t-test was used to determine the statistical significance of the differences between pregnant or lactating rats and the virgin controls. Results and Discussion
The changes in interscapular brown adipose tissue weight, protein and lipid content during pregnancy and lactation are tabulated in Table I. During pregnancy tissue weight increased progressively, reaching its maximum value on day 20 of gestation, whereas it decreased significantly during all the days of lactation studied. The hypertrophy of brown fat during pregnancy was essentially due to the lipid content of the tissue, which increased substantially throughout pregnancy but which was not significantly modified during lactation. These data are compatible with reports on lipid accumulation and higher levels of lipogenesis in brown adipose tissue from pregnant rats [11]. Despite the general hypertrophy of the tissue, there was no increase in the active parts of the tissue throughout pregnancy, as revealed by the unchanged protein content on days 15 and 18 of gestation. In contrast, brown fat protein content was significantly reduced on day 20 of pregnancy and declined progressively during lactation. Its minimum was reached on day 15 of lactation when it was 46% of the virgin control value. The hypotrophy of the active part of the tissue in late pregnant and lactating rats coincides with some studies in mice [9] but differs from that reported by Agius and Williamson [11], where a higher fat-free weight of brown adipose tissue was observed in mid-pregnancy, followed by unchanged
189 TABLE I PROPERTIES OF INTERSCAPULAR BROWN ADIPOSE TISSUE D U R I N G P R E G N A N C Y A N D LACTATION IN THE RAT The values are means_+ S.E. for the number of animals shown in parentheses. * P _< 0.05, ** P _%<0.01, *** P _< 0.001 versus values from virgin rats. Physiological state
Weight (rag)
Protein (mg)
Fat (mg)
Cytochrome oxidase activity
Mitochondrial protein
(t~mol O 2 / m i n per mg protein)
(~mol O 2 / m i n per tissue)
(mg per g tissue protein)
(mg per tissue)
Virgin (8)
332_+15
47.8_+2.7
141_+13
1.13 _+0.02
54.0 + 6.0
551 _+33
26.3 _+2.8
Pregnant 15 days (6) 18days(6) 20days(6)
388_+22* 427_+37 * 453_+24"**
44.0_+2.8 44.4_+3.7 40.2_+1.7"
189_+15" 226_+16 *** 262_+16"**
1.22 + 0.15 1.18 _+0.08 1.08 _+0.07
54.2 _+5.7 52.7 _+9.7 43.0 _+6.0
633 _+92 596 _+52 475 _+47
27.8 +_4.7 26.5 _+3.9 19.1 _+3.0
Lactating 5days(8) 10 days (8) 15days(6)
250_+19"* 243_+12 *** 212_+18"**
32.5_+2.9"* 30.8_+0.9 *** 26.9_+1.9"**
128_+10 119_+ 8 139_+14
0.96_+0.10 0.81 _+0.08 ** 0.88-+0.07 **
31.2_+4.2 ** 24.7_+2.2 ** 24.0-+1.5 **
502_+27 303_+25 *** 354-+50 **
16.3_+2.4 * 9.3_+1.0 *** 9.5-+1.1 ***
values during lactation. Part of this discrepancy may be related to the age of the virgin control rats used, since in the present work they are 3 weeks older than in the Agius and Williamson study that refers to pre-mating values. Cytochrome oxidase activity underwent no significant changes during pregnancy, but the whole tissue activity of the enzyme was substantially reduced on all the days of lactation studied and the specific activity decreased significantly at days 10 and 15 of lactation. The calculated mitochondrial protein content of the tissue showed parallel changes. Thus, it declined significantly only during lactation and, when expressed as a percent of the protein content of the tissue, diminished in 10and 15-day-lactating rats. The profile of changes in G D P binding in brown adipose tissue throughout pregnancy and lactation is depicted in Fig. 1. G D P binding, expressed per mg mitochondrial protein, is not different from virgin controls on all the days of pregnancy studied as well as in 5-day lactating rats. Then, on days 10 and 15 of lactation G D P binding per mg mitochondrial protein fell suddenly to values 44% and 40% of the virgin control ones. Considering the substantial changes in tissue weight and mitochondrial content during pregnancy and lactation described above, the G D P binding per interscapular brown adipose tissue
was calculated since it would be much more relevant biologically; the data are also shown in Fig. 1. G D P binding per tissue was not changed during pregnancy, decreased significantly on day 5 of lactation and fell dramatically in 10- and 15-daylactating rats. To determine whether the large decrease in G D P binding observed in brown fat mitochondria from 10- and 15-day-lactating rats resulted from a change in the number of binding sites or from a change in affinity, Scatchard analysis was performed. This was done in 15-day-lactating rats, and the obtained plots are shown in Fig. 2. The plots were linear over the range of ligand concentrations for both virgin and 15-day-lactating rats, indicating only one type of binding site, in agreement with previous reports [4]. The apparent dissociation constant (KD) was essentially the same for virgin and lactating rats (2.2/~M and 2.3 ~M, respectively) but the maximum binding capacity was decreased from 0.34 nmol per mg mitochondrial protein in virgin rats to 0.21 nmol per mg mitochondrial protein in 15-day-lactating animals. Thus, the decrease in G D P binding found in lactating rats appeared to be due to a reduction in the binding sites without any change in affinity. The consideration of the overall changes in brown adipose tissue throughout the rat breeding cycle described in this paper indicates that brown
190
.H
i i
(a) o P~
0.5.
t t t i
0.15
0.4.
0,10 ,J
L~4 0
0.3-
(D
0.05 0.2"
i i
0.1.
i i i i
0 CQ
0.1
I
i
i
0.2
0.3
0.4
GDP bound
(nmol/mg of protein)
Fig. 2. Scatchard plot of GDP binding to brown adipose tissue
I
(b)
mitochondria from virgin (O) and 15-day-lactating ((3) rats. i
: i 1
4"
2.
I
15 VIRGIN
18
20
5
10
PREGNANCY
LACTATION
(Days)
(Days)
15
Fig. l . Changes in GDP binding to interscapular brown adipose tissue throughout pregnancy and lactation in the rat. (a) G D P binding per mg mitochondrial protein; (b) G D P binding per total tissue. The results are means+ S.E. (indicated by bars) for 6 - 8 rats at each day. * P < 0.05, ** P_< 0.01, *** P_< 0.001 compared with values for virgin controls.
adipose tissue oxidative capacity and thermogenic activity are essentially unchanged during pregnancy, whereas there is a progressive reduction in brown fat thermogenic function during lactation. Even though no previous data are available on brown adipose tissue cytochrome oxidase activity and GDP binding during pregnancy and early stages of lactation in the rat, the present results agree with studies in mid-lactating rats [16], but differ from reports in late-pregnant mice, as they show a decrease in both cytochrome oxidase activity and mitochondrial GDP binding [9], although
this discrepancy may be explained on the basis of the species difference. The suppression of brown fat thermogenic activity during the breeding cycle has been claimed to be a significant energy-sparing mechanism to improve the energy efficiency, related to the nutritional stress caused by conceptus maintenance and milk synthesis. Considering that the total conceptus weight usually accounts for a higher percent of the total maternal weight in late-pregnant mice than in rats, it might be expected that the associated nutritional challenge was greater in mice, and thus explain the earlier inactivation of brown fat in the mouse breeding cycle. The progressive reduction of brown adipose tissue activity during lactation agrees with the progressive increase in the need for milk production as lactation proceeds. The decrease in brown adipose tissue activity develops progressively during lactation, involving different stages that show a striking sequential appearance as lactation goes on. In late pregnancy and early lactation, tissue protein a n d its mitochondrial component are lowered, and the overall decrease in the thermogenic activity of brown fat in the first days of lactation is attained through the lower mitochondrial content of the tissue. Only later, in mid-lactation, when the amount of tissue and its mitochondrial component are minimal, does the GDP binding per mitochondriai protein decrease substantially. The timing of changes in brown adipose tissue
191 in a physiological a d a p t a t i o n to low activity such as lactation contrasts with the sequence of events occurring in situations of b r o w n adipose tissue activation such as, for example, cold acclimatization, that is k n o w n to involve two kinds of events; an initial increase in the m i t o c h o n d r i a l p r o t o n c o n d u c t a n c e p a t h w a y activity, followed by a general hypertrophic response a n d m i t o c h o n d r i a l proliferation [4]. However, the p a t t e r n of sequential changes observed d u r i n g the b r e e d i n g cycle resembles much more those described in other situations of reduction in b r o w n fat activity, such as starvation. I n this sense, a decrease in the m i t o c h o n d r i a l p r o t e i n c o n t e n t of the tissue appears in the first days of fasting [17], whereas a reduction in G D P b i n d i n g has been reported only after longer periods of starvation [18]. These findings suggest that, in situations in which b r o w n adipose tissue progressively reduces its activity, it is the m i t o c h o n d r i a l rather than m i t o c h o n d r i a l G D P b i n d i n g , c o n t e n t of the tissue, which is highly susceptible to modification.
References 1 Rothwell, N.J. and Stock, M.J. (1979) Nature (Lond.) 281, 31-35
2 Foster, D.O. and Frydman, M.L. (1978) Can. J. Physiol. Pharmacol. 56, 110-122 3 Nicholls, D.G, (1976) Eur. J. Biochem. 62, 223-228 4 Sundin, U. and Cannon, B. (1980) Comp. Biochem. Physiol. 65B, 463-471 5 Himms-Hagen, J., Triandfillou, J. and Gwilliam, C. (1981) Am. J. Physiol. 241, El16-E120 6 Goodbody, A.E. and Trayhurn, P. (1981) Biochem. J. 194, 1019-1022 7 Holt, S. and York, D.A. (1982) Biochem. J. 208, 819-822 8 Trayhurn, P., Jones, P.M., McGuckin, M.M. and Goodbody, A.E. (1982a) Nature (Lond.) 295, 323-325 9 Trayhurn, P., Douglas, J.B. and McGuckin, M.M. (1982b) Nature (Lond.) 298, 59-60 10 Cooney, G.J. and Newsholme, E.A. (1984) Trends Biochem. Sci. 9, 303-305 11 Agius, L. and Williamson, D.H. (1980) Biochem. J. 190, 477-482 12 Folch, J., Lees, M. and Sloane-Stanley,G.H. (1957) J. Biol. Chem. 226, 497-509 13 Cannon, B. and Lindberg, O. (1979) Methods Enzymol. 55, 65-78 14 Wang, S. and Smith, L.R. (1975) Anal. Biochem. 63, 414-417 15 Rafael, J. (1983) Methods of Enzymatic Analysis (H.U. Bergmeyer, ed.), Vol. 3, pp. 266-283, Verlag Chemic, Basel 16 Isler, D., Trayhurn, P. and Lunn, P.G. (1984) Ann. Nutr. Metab. 28, 101-109 17 Desautels, M. (1985) Am. J. Physiol. 249, E99-E106 18 Rothwell, N.J., Saville, M.E. and Stock, M.J. (1984) Biosci. Rep. 4, 351-357
882 (1986) 187-191
187
Elsevier BBA22341
S e q u e n t i a l c h a n g e s in b r o w n a d i p o s e t i s s u e c o m p o s i t i o n , c y t o c h r o m e o x i d a s e a c t i v i t y a n d G D P b i n d i n g t h r o u g h o u t p r e g n a n c y a n d l a c t a t i o n in t h e r a t Francesc Villarroya, Antonio Felipe and Teresa Mampel
*
C,~tedra de Fisiologia General de la Facultat de Biologia, Unioersitat de Barcelona, Avinguda Diagonal 645, 08071 Barcelona (Spain)
(ReceivedJanuary20th, 1986)
Key words: Cytochromeoxidase; GDP binding; Lipid composition; Pregnancy; Lactation; (Rat brown adipose) The sequential appearance of changes in interscapular brown adipose tissue composition, cytochrome oxidase activity and GDP binding was studied throughout pregnancy and lactation in the rat. Brown adipose tissue was hypertrophied during pregnancy because of progressive lipid accumulation, whereas its mitochondrial component and G D P binding to brown fat mitochondria were unchanged. In early lactation (day S) there was a decrease in the overall GDP binding to brown fat only because of the lower mitochondrial protein content. In late stages of lactation (days 10 and IS), the amount of tissue and its mitochondrial protein content were minimal and the G D P binding per mitochondrial protein decreased substantially. Scatchard analysis in day-IS-lactating rats indicated a large decrease in G D P binding sites without any changes in affinity. It is concluded that the diminished thermogenic activity of brown fat in lactation is attained through changes at different structural levels of the tissue occurring in a characteristic sequential trend; first a reduction in its mitochondrial component, and only later, at mid-lactation, a decrease in the specific mitochondrial proton conductance pathway activity.
Introduction
Brown adipose tissue has been recognized as the main site for both diet [1] and cold-induced [2] non-shivering thermogenesis in rodents. Thermogenesis in brown fat is due to the proton conductance pathway [3] that allows protons to translocate through the inner mitochondrial membrane without coupling to ATP synthesis. The activity of this pathway can be measured by determining the specific binding of G D P to brown adipose tissue mitochondria, due to the property of purine nucleotides to bind to a M r 32 000 mitochondrial protein inhibiting the proton conductance pathway. The high thermogenic activity of brown adipose * To whom correspondenceshould be addressed.
tissue in the cold-acclimated or chronically overfed animals is achieved through increases in the activity of the mitochondrial proton conductance pathway together with an increment in the mitochondria content and a general hypertrophy a n d / o r hyperplasia of the tissue [4,5]. On the other hand, brown fat thermogenesis has been reported to be impaired in animals with lowered energy expenditure and subsequently obese, such as the o b / o b and d b / d b mice and the f a / f a rat. The defective brown adipose thermogenic activity in these animals is associated with a diminished mitochondrial G D P binding [6-8], and a decrease in the mitochondrial component of the tissue has also been reported in the o b / o b [8] and d b / d b mice [6]. Much less is known about functional modifications in the brown adipose tissue under physio-
0304-4165/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
188 logical situations associated with substantial changes in energy intake and energy expenditure such as those of the breeding cycle. Trayhurn et al. [9] have described diminished activity of the proton conductance pathway in brown fat from pregnant and lactating mice, together with a decrease in the oxidative capacity of the tissue assessed by the cytochrome oxidase activity. However, studies on lipogenesis, considered by some authors to be closely linked to thermogenesis in brown fat [10] have shown greater lipogenesis and increased fatfree mass in the tissue from pregnant rats, as well as diminished lipogenesis without signs of hypotrophy in the mid-lactating rat [11]. The aim of the present work is to study further the changes in brown adipose tissue composition and thermogenic activity during pregnancy and lactation by means of determining the appearance in time of brown fat changes throughout the different stages of the breeding cycle in the rat. Materials and Methods
Female Wistar rats with an initial weight of 210-220 g were used. They were mated with adult males and the day of pregnancy was determined by the presence of spermatozoa in vaginal smears. After fecundation the rats were kept in individual cages, and when lactating rats were studied litter sizes were adjusted to ten pups. The animals were maintained under automatically controlled temperature conditions (23 + 1 °C) and 12-h light-dark cycles. They were fed ad libitum on a stock diet (A04 Panlab, Barcelona, Spain). All experiments were started between 09:00 and 10:00 h. Pregnant rats of 15, 18 and 20 days of gestation, lactating rats 5, 10 and 15 days after parturition and virgin controls were killed by decapitation. The interscapular brown adipose tissue was rapidly removed and dissected free of any adhering connective tissue or white adipose tissue. It was weighed and a small sample was used for lipid determination carried out as described by Folch et al. [12]. The remaining tissue was homogenized in 0.25 M sucrose, and mitochondria were prepared as described by Cannon and Lindberg [13]. The purine nucleotide binding assay was performed at room temperature (22°C) using [8-3H]GDP (Amersham, U.K.) using the method of Nicholls
[3] as modified by Goodbody and Trayhurn [6]. Scatchard analysis of [3H]GDP binding was performed as described above with concentrations ranging from 0.6 to 20 ~M. Nonspecific binding, determined by the presence of 100 #M GDP, was negligible. The protein content of mitochondrial preparations and the original tissue homogenate was determined [14] and the cytochrome oxidase activity (EC 1.9.3.1) was measured by a polarographic method [15]. The recovery of cytochrome oxidase activity in the mitochondrial preparations was calculated in each sample and its value was used to estimate the amount of mitochondrial protein in the interscapular brown adipose tissue. Student's t-test was used to determine the statistical significance of the differences between pregnant or lactating rats and the virgin controls. Results and Discussion
The changes in interscapular brown adipose tissue weight, protein and lipid content during pregnancy and lactation are tabulated in Table I. During pregnancy tissue weight increased progressively, reaching its maximum value on day 20 of gestation, whereas it decreased significantly during all the days of lactation studied. The hypertrophy of brown fat during pregnancy was essentially due to the lipid content of the tissue, which increased substantially throughout pregnancy but which was not significantly modified during lactation. These data are compatible with reports on lipid accumulation and higher levels of lipogenesis in brown adipose tissue from pregnant rats [11]. Despite the general hypertrophy of the tissue, there was no increase in the active parts of the tissue throughout pregnancy, as revealed by the unchanged protein content on days 15 and 18 of gestation. In contrast, brown fat protein content was significantly reduced on day 20 of pregnancy and declined progressively during lactation. Its minimum was reached on day 15 of lactation when it was 46% of the virgin control value. The hypotrophy of the active part of the tissue in late pregnant and lactating rats coincides with some studies in mice [9] but differs from that reported by Agius and Williamson [11], where a higher fat-free weight of brown adipose tissue was observed in mid-pregnancy, followed by unchanged
189 TABLE I PROPERTIES OF INTERSCAPULAR BROWN ADIPOSE TISSUE D U R I N G P R E G N A N C Y A N D LACTATION IN THE RAT The values are means_+ S.E. for the number of animals shown in parentheses. * P _< 0.05, ** P _%<0.01, *** P _< 0.001 versus values from virgin rats. Physiological state
Weight (rag)
Protein (mg)
Fat (mg)
Cytochrome oxidase activity
Mitochondrial protein
(t~mol O 2 / m i n per mg protein)
(~mol O 2 / m i n per tissue)
(mg per g tissue protein)
(mg per tissue)
Virgin (8)
332_+15
47.8_+2.7
141_+13
1.13 _+0.02
54.0 + 6.0
551 _+33
26.3 _+2.8
Pregnant 15 days (6) 18days(6) 20days(6)
388_+22* 427_+37 * 453_+24"**
44.0_+2.8 44.4_+3.7 40.2_+1.7"
189_+15" 226_+16 *** 262_+16"**
1.22 + 0.15 1.18 _+0.08 1.08 _+0.07
54.2 _+5.7 52.7 _+9.7 43.0 _+6.0
633 _+92 596 _+52 475 _+47
27.8 +_4.7 26.5 _+3.9 19.1 _+3.0
Lactating 5days(8) 10 days (8) 15days(6)
250_+19"* 243_+12 *** 212_+18"**
32.5_+2.9"* 30.8_+0.9 *** 26.9_+1.9"**
128_+10 119_+ 8 139_+14
0.96_+0.10 0.81 _+0.08 ** 0.88-+0.07 **
31.2_+4.2 ** 24.7_+2.2 ** 24.0-+1.5 **
502_+27 303_+25 *** 354-+50 **
16.3_+2.4 * 9.3_+1.0 *** 9.5-+1.1 ***
values during lactation. Part of this discrepancy may be related to the age of the virgin control rats used, since in the present work they are 3 weeks older than in the Agius and Williamson study that refers to pre-mating values. Cytochrome oxidase activity underwent no significant changes during pregnancy, but the whole tissue activity of the enzyme was substantially reduced on all the days of lactation studied and the specific activity decreased significantly at days 10 and 15 of lactation. The calculated mitochondrial protein content of the tissue showed parallel changes. Thus, it declined significantly only during lactation and, when expressed as a percent of the protein content of the tissue, diminished in 10and 15-day-lactating rats. The profile of changes in G D P binding in brown adipose tissue throughout pregnancy and lactation is depicted in Fig. 1. G D P binding, expressed per mg mitochondrial protein, is not different from virgin controls on all the days of pregnancy studied as well as in 5-day lactating rats. Then, on days 10 and 15 of lactation G D P binding per mg mitochondrial protein fell suddenly to values 44% and 40% of the virgin control ones. Considering the substantial changes in tissue weight and mitochondrial content during pregnancy and lactation described above, the G D P binding per interscapular brown adipose tissue
was calculated since it would be much more relevant biologically; the data are also shown in Fig. 1. G D P binding per tissue was not changed during pregnancy, decreased significantly on day 5 of lactation and fell dramatically in 10- and 15-daylactating rats. To determine whether the large decrease in G D P binding observed in brown fat mitochondria from 10- and 15-day-lactating rats resulted from a change in the number of binding sites or from a change in affinity, Scatchard analysis was performed. This was done in 15-day-lactating rats, and the obtained plots are shown in Fig. 2. The plots were linear over the range of ligand concentrations for both virgin and 15-day-lactating rats, indicating only one type of binding site, in agreement with previous reports [4]. The apparent dissociation constant (KD) was essentially the same for virgin and lactating rats (2.2/~M and 2.3 ~M, respectively) but the maximum binding capacity was decreased from 0.34 nmol per mg mitochondrial protein in virgin rats to 0.21 nmol per mg mitochondrial protein in 15-day-lactating animals. Thus, the decrease in G D P binding found in lactating rats appeared to be due to a reduction in the binding sites without any change in affinity. The consideration of the overall changes in brown adipose tissue throughout the rat breeding cycle described in this paper indicates that brown
190
.H
i i
(a) o P~
0.5.
t t t i
0.15
0.4.
0,10 ,J
L~4 0
0.3-
(D
0.05 0.2"
i i
0.1.
i i i i
0 CQ
0.1
I
i
i
0.2
0.3
0.4
GDP bound
(nmol/mg of protein)
Fig. 2. Scatchard plot of GDP binding to brown adipose tissue
I
(b)
mitochondria from virgin (O) and 15-day-lactating ((3) rats. i
: i 1
4"
2.
I
15 VIRGIN
18
20
5
10
PREGNANCY
LACTATION
(Days)
(Days)
15
Fig. l . Changes in GDP binding to interscapular brown adipose tissue throughout pregnancy and lactation in the rat. (a) G D P binding per mg mitochondrial protein; (b) G D P binding per total tissue. The results are means+ S.E. (indicated by bars) for 6 - 8 rats at each day. * P < 0.05, ** P_< 0.01, *** P_< 0.001 compared with values for virgin controls.
adipose tissue oxidative capacity and thermogenic activity are essentially unchanged during pregnancy, whereas there is a progressive reduction in brown fat thermogenic function during lactation. Even though no previous data are available on brown adipose tissue cytochrome oxidase activity and GDP binding during pregnancy and early stages of lactation in the rat, the present results agree with studies in mid-lactating rats [16], but differ from reports in late-pregnant mice, as they show a decrease in both cytochrome oxidase activity and mitochondrial GDP binding [9], although
this discrepancy may be explained on the basis of the species difference. The suppression of brown fat thermogenic activity during the breeding cycle has been claimed to be a significant energy-sparing mechanism to improve the energy efficiency, related to the nutritional stress caused by conceptus maintenance and milk synthesis. Considering that the total conceptus weight usually accounts for a higher percent of the total maternal weight in late-pregnant mice than in rats, it might be expected that the associated nutritional challenge was greater in mice, and thus explain the earlier inactivation of brown fat in the mouse breeding cycle. The progressive reduction of brown adipose tissue activity during lactation agrees with the progressive increase in the need for milk production as lactation proceeds. The decrease in brown adipose tissue activity develops progressively during lactation, involving different stages that show a striking sequential appearance as lactation goes on. In late pregnancy and early lactation, tissue protein a n d its mitochondrial component are lowered, and the overall decrease in the thermogenic activity of brown fat in the first days of lactation is attained through the lower mitochondrial content of the tissue. Only later, in mid-lactation, when the amount of tissue and its mitochondrial component are minimal, does the GDP binding per mitochondriai protein decrease substantially. The timing of changes in brown adipose tissue
191 in a physiological a d a p t a t i o n to low activity such as lactation contrasts with the sequence of events occurring in situations of b r o w n adipose tissue activation such as, for example, cold acclimatization, that is k n o w n to involve two kinds of events; an initial increase in the m i t o c h o n d r i a l p r o t o n c o n d u c t a n c e p a t h w a y activity, followed by a general hypertrophic response a n d m i t o c h o n d r i a l proliferation [4]. However, the p a t t e r n of sequential changes observed d u r i n g the b r e e d i n g cycle resembles much more those described in other situations of reduction in b r o w n fat activity, such as starvation. I n this sense, a decrease in the m i t o c h o n d r i a l p r o t e i n c o n t e n t of the tissue appears in the first days of fasting [17], whereas a reduction in G D P b i n d i n g has been reported only after longer periods of starvation [18]. These findings suggest that, in situations in which b r o w n adipose tissue progressively reduces its activity, it is the m i t o c h o n d r i a l rather than m i t o c h o n d r i a l G D P b i n d i n g , c o n t e n t of the tissue, which is highly susceptible to modification.
References 1 Rothwell, N.J. and Stock, M.J. (1979) Nature (Lond.) 281, 31-35
2 Foster, D.O. and Frydman, M.L. (1978) Can. J. Physiol. Pharmacol. 56, 110-122 3 Nicholls, D.G, (1976) Eur. J. Biochem. 62, 223-228 4 Sundin, U. and Cannon, B. (1980) Comp. Biochem. Physiol. 65B, 463-471 5 Himms-Hagen, J., Triandfillou, J. and Gwilliam, C. (1981) Am. J. Physiol. 241, El16-E120 6 Goodbody, A.E. and Trayhurn, P. (1981) Biochem. J. 194, 1019-1022 7 Holt, S. and York, D.A. (1982) Biochem. J. 208, 819-822 8 Trayhurn, P., Jones, P.M., McGuckin, M.M. and Goodbody, A.E. (1982a) Nature (Lond.) 295, 323-325 9 Trayhurn, P., Douglas, J.B. and McGuckin, M.M. (1982b) Nature (Lond.) 298, 59-60 10 Cooney, G.J. and Newsholme, E.A. (1984) Trends Biochem. Sci. 9, 303-305 11 Agius, L. and Williamson, D.H. (1980) Biochem. J. 190, 477-482 12 Folch, J., Lees, M. and Sloane-Stanley,G.H. (1957) J. Biol. Chem. 226, 497-509 13 Cannon, B. and Lindberg, O. (1979) Methods Enzymol. 55, 65-78 14 Wang, S. and Smith, L.R. (1975) Anal. Biochem. 63, 414-417 15 Rafael, J. (1983) Methods of Enzymatic Analysis (H.U. Bergmeyer, ed.), Vol. 3, pp. 266-283, Verlag Chemic, Basel 16 Isler, D., Trayhurn, P. and Lunn, P.G. (1984) Ann. Nutr. Metab. 28, 101-109 17 Desautels, M. (1985) Am. J. Physiol. 249, E99-E106 18 Rothwell, N.J., Saville, M.E. and Stock, M.J. (1984) Biosci. Rep. 4, 351-357