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Reduced noradrenaline turnover in brown adipose tissue of lactating rats
Camp. Biochem. Physiol. Vol. 86A, No. 3, pp. 481483,
0300-9629187 $3.00+ 0.00 0 1987 Pergamon Journals Ltd
1987
Printed in Great Britain
REDUCED NORADRENALINE TURNOVER IN BROWN ADIPOSE TISSUE OF LACTATING RATS FRANCESC VILLARROYA, ANTONIO FELIPE and TERESA MAMPEL Fisiologia General, Facultat de Biologia, Universitat de Barcelona, 08071 Barcelona, Spain (Received 22 May 1986) Abstract-l. Brown adipose tissue properties as well as noradrenaline turnover in the tissue were determined in 15-day lactating rats and virgin controls. 2. Brown adipose tissue thermogenic activity was reduced in lactating rats as shown by a decrease in weight, cytochrome oxidase activity and mitochondrial GDP-binding. 3. The noradrenaline turnover rate was lower in brown adipose tissue from lactating rats. 4. It is suggested that diminished sympathetic activity in brown adipose tissue may be a major cause of the reduced tissue thermogenic activity during lactation.
activity in the tissue by measuring the rate of noradrenaline turnover in brown adipose tissue from mid-lactating rats.
INTRODUCTION Brown adipose tissue shivering thermogenesis Frydman, 1978) and
is the major site for nonresponse to cold (Foster and overfeeding (Rothwell and
Stock, 1979) in rodents. The mechanism responsible for thermogenesis in brown fat is considered to be the mitochondrial proton conductance pathway described by Nicholls (1974). Thus, chronically cold+xposed rats show a high activity of the proton conductance pathway as well as an increase in the tissue size and mitochondrial content (Sundin and Cannon, 1980), and similar responses are observed in the tissue from chronically overfed rodents (Himms-Hagen et al., 1981). Brown fat thermogenesis has been reported to be suppressed in mid-lactating mice (Trayhurn et al., 1982) and rats (Villarroya et al., 1986) despite the hyperphagia that characterizes this physiological period. It has been suggested that diminished brown adipose tissue thermogenic activity during lactation would be an energy-sparing adaptation advantageous to the dam because of the nutritional stress linked to milk production. However, little is known about the regulatory mechanisms involved in this reduction of brown adipose tissue function. The sympathetic nervous system seems to play a key role in the regulation of brown adipose tissue activity. Thus, the sympathetic activity in brown fat-assessed from the rate of noradrenaline turnover-is increased in cold-exposed as well as in chronically-overfed rats (Young et al., 1982) and most of the brown adipose tissue modifications due to chronic cold exposure or overfeeding can be mimicked by noradrenaline treatment (Mory et al., 1984). The present work was undertaken to determine whether the reduced brown fat thermogenesis during lactation is related to changes in the sympathetic
Correspondence should be sent to Dr. Teresa Fisiologia Barcelona,
Mampel, General, Facultat de Biologia, Universitat de Avda Diagonal 645,08071 Barcelona, Spain. 481
MATERIALS AND METHODS Female Wistar rats (Rattus norvegicus) fed ad libitum Purina chow pellets (A04 type, Panlab, Barcelona) were mated with adult males when weighing 210-220 g. They were caged individually and maintained in a temperaturecontrolled environment (21 f 1°C). After parturition all litters were adjusted to 10 pups. Dams were used 15 days after parturition and compared with age-matched virgin controls. After decapitation of virgin and lactating rats the interscapular brown adipose tissue was removed, weighed and a small sample used for total lipid determination (Folch et al., 1957). Mitochondria were isolated from the remaining tissue (Cannon and Lindberg, 1979) and incubated for IO min with 10 PM (jH)-GDP essentially as described by Nicholls (1974) for the GDP-binding assay. Protein content of the mitochondrial preparations and the original tissue homogenates were determined (Wang and Smith, 1975) as well as the cytochrome oxidase activity (EC 1.9.3.1.) that was measured polarographically (Rafael, 1983). The recovery of cytochrome oxidase activity did not differ significantly between the two groups and the individual values were used to estimate the amount of mitochondrial protein in the interscapular brown adipose tissue and, subsequently, the total mitochondrial GDP-binding in the tissue. Statistical comparisons of the properties in brown fat from lactating and virgin control rats were performed with the Student’s t-test. Noradrenaline turnover was estimated in another set of 15-day lactating dams and virgin controls by measuring the noradrenaline concentration in interscapular brown adipose tissue at 0, 3 and 6 hr after i.p. injection of 80 mg/kg of a-methyl-p-tyrosine (Sigma, USA) as previously described (Spector et al., 1965; Young and Landsberg, 1977). After decapitation of the animals, interscapular brown adipose tissue was rapidly removed, weighed, homogenized in chilled 0.1 N perchloric acid containing. 0.1 mM reduced glutathione, and centrifuged at 0°C. Aliiuots of the supernatants were analyzed for noradrenaline content with a high performance liquid chromatography method (Eriksson and Persson, 1982) using a ~Bondapak C-18 column (Waters). The mobile phase was 0.04 M citrate/O. 1 M acetate buffer (PH 5.2) containing 10% methanol and 5 mM octanosulfonic acid. A flow rate of 1.5 ml/min was used and
482
FRANCESC VILLARROYA et al.
noradrenaline content was quantified with an electrochemical detector. Noradrenaline concentration data were set in a semi-
2000 c
logarithmic plot against time and the slope (fractional turnover rate, k) of the graph was calculated by the least squares method. Comparison of the fractional turnover rates was made using analysis of covariance. Noradrenaline turnover rates were calculated as the product of the fractional turnover rate and the endogenous noradrenaline concentration at the zero point. Ninety-five confidence intervals were determined for the noradrenaline turnover rates as described (Taubin et al., 1972).
RESULTS AND DISCUSSION
Composition, cytochrome oxidase activity and GDP-binding of interscapular brown adipose tissue from 15-day lactating rats compared with virgin controls are presented in Table 1. Interscapular brown fat weight was reduced in lactating rats to 69% of that of age-matched virgin controls. Tissue lipid content was not significantly altered by lactation whereas the protein content of interscapular brown adipose tissue from lactating rats was reduced. Both cytochrome oxidase specific activity, as well as whole tissue activity, were lower in lactating rats than in controls. Specific mitochondrial GDP-binding (nmol GDP/mg mitochondrial protein) was also significantly lower in dams and total mitochondrial GDP-binding (nmol GDP/interscapular brown fat) was reduced to 20% of that of virgin controls. These results indicate a generalized decrease in the activity of brown adipose tissue during lactation, essentially in agreement with previous reports in mid-lactating rats (Isler et al., 1984; Villarroya et al., 1986) and mice (Trayhurn et al., 1982). This low brown fat activity is due to the hypotrophy of the tissue, a decrease in its overall oxidative capacity-as shown by the cytochrome oxidase data-and a reduction in the mitochondrial proton conductance pathway activity estimated by the GDP-binding method. Data on noradrenaline content and turnover in interscapular brown adipose tissue from 15-day lactating rats are shown in Table 2. The slopes of noradrenaline concentration decay graphs are presented in Fig. 1. Noradrenaline concentrations
0
6
3
TIME
(hours)
Fig. 1. Noradrenaline turnover in interscapular brown adipose tissue from virgin and 1S-day lactating rats. All data are plotted as mean + SEM for endogenous noradrenaline in interscapular brown adipose tissues from 5 animals in each group at 0, 3 and 6 hr after the injection of a-methyl-p-tyrosine (80 mg/kg, i.p.). Closed circles represent virgin controls and open circles 15day lactating rats.
Virgin
Lactating
476.0 f 32.1
~- 330.1 + 28.lt
Lipid content (mg) per g of tissue per tissue
420.3 f 30.4 202.1 i 19.2
561.1 + 15.6: 185.0 f 18.2
were higher in interscapular brown adipose tissue from lactating dams but the total tissue noradrenaline content was not significantly changed. The fractional noradrenaline turnover rate (k) in the tissue was significantly reduced in lactating rats (an average 33% of that of virgin controls) while noradrenaline turnover was also significantly slower in the interscapular brown adipose tissue from dams (a mean 43% of that of virgin controls). The finding that noradrenaline turnover is reduced in interscapular brown fat from mid-lactating rats indicates that sympathetic activity in the tissue is reduced in this situation. Noradrenaline has been described to have a key role in the regulation of brown adipose tissue composition and activity (Desautels and Himms-Hagen, 1979; Mory et al., 1984) and our results suggest that low sympathetic nervous system activity in the tissue may have a major responsibility in the changes in brown fat described below leading to its lowered activity during lactation. Sympathetic activity in brown adipose tissue is considered to be closely related to food consumption (Landsberg and Young, 1983). Thus, fasted rats show reduced brown fat thermogenesis (Rothwell et al., 1984) associated with diminished sympathetic activity in the tissue (Young et al., 1982) whereas opposite changes take place in brown adipose tissue from overfed rats (Young et al., 1982). However, reduced brown adipose tissue thermogenesis and nor-
Protein content (mg) per g of tissue per tissue
144.0 + 6.1 67.5 +_ 3.9
122.1 f6.4’ 40.1 f 2.9:
Table 2. Noradrenaline content and turnover in interscapular brown adipose tissue from virgin and l5-day lactating rats
Table
Weight
I. Properties
of interscapular brown adipose tissue from virgin and I S-day lactating rats
(mg)
Cytochrome
Virgin
oxidase activity
(ymol O,/min) per mg of tissue protein per tissue GDP-binding (nmol GDP) per mg of mitochondrial protein per tissue
3.9 t 0.2 261.3 + 21.4
0.38 f 0.03 8.01 k 0.79
2.9 f 0.2t 119.7 * IO.71
0. I6 f 0.031 1.60fO.l3$
Results are means f SEM of 68 different animals. Statistical significance of the comparisons between virgin and lactating rats: ‘P 5 0.05; tP < 0.01; IP 5 0.001.
Noradrenaline (rig/g tissue) Noradrenaline (ngitissue) Noradrenaline fractional turnover rate, k (%/hr) Noradrenaline turnover (rig/g tissue/hr)
Lactating
1345 _t II2 646 + 90
1714 f 24* 512 & 45
5.1 + 0.5
I .8 + 0.6t
69. I f 5.7
30.0 I8.4t
Results are means + SEM of 5 animals for noradrenaline content data as well as for each time used to obtain the turnover data. Statistical significance of the comparisons between virgin and lactating rats: lP 5 0.05, tP 5 0.01.
Reduced NA turnover in BAT of lactating rats
adrenaline turnover in the tissue are associated with increased food consumption in pathological states such as the genetically obese (ob/ob) mice (Knehans and Romsos, 1982). Our results indicate that a reduction in the sympathetic activity in brown fat may also be the m~hanism responsible for the reduction in brown adipose tissue activity during lactation despite the associated hyperphagia. The mechanism for decreased thermogenesis in pathological states seems thus to be the same as in our physiological situation, suggesting a unity in the mechanisms responsible for brown fat modifications. On the other hand, even though the peripheral signals modulating sympathetic activity in relation to food intake are still not well known, circulating glucose and insulin have been claimed to be involved in this role (Landsberg and Young, 1983). In this sense, the low glucose and insulin levels occurring in the mid-lactating rat (Flint ef al., 1979; Burn01 et al., 1983) would be consistent with the reduced sympathetic activity found in brown fat in this situation. In summary, present results indicate that sympathetic activity, assessed by means of noradrenaline turnover, is reduced in brown adipose tissue from mid-lactating rats, in good correlation with a lower oxidative and thermogenic activity of the tissue in this situation. This finding suggests that low sympathetic nervous system activity in brown fat may be a major factor leading to its reduced thermogenic activity during lactation. Acknowledgemenr-The authors wish to express their gratitude to Maria Jose Sarrias from the Laboratory of Neurochemistry (C.S.I.C., Barcelona) for her help in the analysis of noradrenaline.
REFERENCES Burn01 A. F., Leturque A., Ferre P. and Girard J. (1983) Glucose metabolism during lactation in the rat: quantitative and regulatory aspects. Am. J. Physioi. 245, E351-E358. Cannon B. and Lindberg 0. (1979) Mitochondria from brown adipose tissue: isolation and properties. In Merhads in Enzymology (Edited by Fleischer S. and Packer L.) pp. 65-79. Academic Press, New York. Desautels M. and Himms-Hagen J. (1979) Roles of noradrenaline and protein synthesis in the cold-induced increase in purine nucleotide binding by rat brown adioose tissue mitochondria. Can. J. Phvsiol. 57.968-976. Eriksion B. M. and Persson B. A. (1982)Determination of catecholamines in rat heart tissue and plasma samples by liquid chromatography with electrochemical detector. J. Chromarogr. 228, 143- 154. Flint D. J., Sinnett-Smith P. A., Clegg R. A. and Vernon R. 6. (1979) Role of insulin receptors in the changing metabolism of adipose tissue during pregnancy and lactation in the rat. &hem. J. 182, 421-4>7. Folch J., Lees M. and Sloane-Stanley G. H. (1957) A simple method for the isolation and purification of the total lipids from animal tissues. J. biol. Chem. 226, 497-509.
483
Foster D. 0. and Frydman M. L. (1978) Non-shivering thermogenesis in the rat. II. Measurements of blood flow with microspheres point to brown adipose tissue as the dominant site of calorigenesis induced by noradrenaline. Can .J. Physiol. Pharmac. 56, 110-122.
Himms-Hagen J., Triand~llou J. and Gwiiliam C. (1981) Brown adipose tissue of cafeteria-fed rats. Am. J. Phy.~i~~/. 241, El 16E120. lsler D., Trayhurn P. and Lunn P. G. (I 984) Brown adipose tissue metabolism in lactating rats: the effect of litter size. Ann. Nutr. Metab. 28, 101-109.
Knehans A. W. and Romsos D. R. (1982) Reduced norepinephrine turnover in brown adipose tissue of the ob,/ob mice. Am. J. Ph.tziui. 242, E2533E261. Landsberg L. and Young J. B. (1983) Autonomic regulation of thermogenesis. in Mammalian Thermogenesis (Edited by Girardier L. and Stock M. J.) pp. 999140. Chapman and Hall, London. Mory G., Bouillaud F., Combes-George M. and Ricquier D. (1984) Noradrenaline controls the concentration of the uncoupling protein in brown adipose tissue. FEBS Lrtt. 166, 393-396. Nicholls D. G. (1974) Hamster brown adipose tissue mitochondria. The control of respiration and the proton electrochemical potential gradient by possible physiological effecters of the proton conductance of the inner membrane. Eur. J. Biochem. 37, 523 530. Rafael J, (1983) Cytochrome ox&se. In Methods q/ Enzymafic Analysi.s. (Edited by Bergmeyer H. V.), Vol 3. pp. 266283. Verlag Chimie, Base]. Rothwell N. J. and Stock M. J. (1979) A role for brown adipose tissue in diet-induced thermogenesis. Nururr (Lo&.) 281, 31-35. Rothwell N. J., Saville E. and Stock M. J. (1984) Brown fat activity in fasted and re-fed rats, Biosci. Rep. 4, 35 I 357. Spector S., Sjoerdsma A. and Udenfriend S. (1965) Blockade of endogenous norepinephrine synthesis by atphamethvhyrosine, an inhibitor of tyrosine hydroxylase. J. Pharmac. exp. Ther. 141, 8695.
Sundin U. and Cannon B. (1980) GDP-binding to the brown fat mitochondria of heveloping and coldyadapted rats. Camp. Biochem. Physiol. 65B, 463-481. Taubin H. L.. Djahanguiri B. and Landsberg L. (1972) Noradrenaline concentration and turnover in different regions of the gastrointestinal tract of the rat: an approach to the evaluation of sympathetic activity in the gut. Gut 13, 790-795. Trayhum P., Douglas J. B. and McGuckin M. M. (1982) Brown adipose tissue thermogenesis is “suppressed” during lactation in mice. Nature (Lord.) 298, 594. Villarroya F., Feiipe A. and Mampel T. (1986) Sequential changes in brown adipose tissue composition, cytochrome oxidase activity and GDP-binding throughout pregnancy and lactation in the rat. Biochim. biophys. Ada. (in press). Wang S. and Smith L. R. (1975) Lowry determination of protein in the presence of Triton X-100. Analyr. Biochem. 63, 414-417.
Young J. B. and Landsberg L. (1977) Stimulation of the sympathetic nervous system during sucrose feeding. Nature (Land.) 269, 61S-61 7. Young J. B., Saville E., Rothwell N. J., Stock M. J. and Landsberg L. (1982) Effect of diet and cold exposure on norepinephrine turnover in brown adipose tissue of the rat. J. clin. Inrest. 69, 1061- 1071.
0300-9629187 $3.00+ 0.00 0 1987 Pergamon Journals Ltd
1987
Printed in Great Britain
REDUCED NORADRENALINE TURNOVER IN BROWN ADIPOSE TISSUE OF LACTATING RATS FRANCESC VILLARROYA, ANTONIO FELIPE and TERESA MAMPEL Fisiologia General, Facultat de Biologia, Universitat de Barcelona, 08071 Barcelona, Spain (Received 22 May 1986) Abstract-l. Brown adipose tissue properties as well as noradrenaline turnover in the tissue were determined in 15-day lactating rats and virgin controls. 2. Brown adipose tissue thermogenic activity was reduced in lactating rats as shown by a decrease in weight, cytochrome oxidase activity and mitochondrial GDP-binding. 3. The noradrenaline turnover rate was lower in brown adipose tissue from lactating rats. 4. It is suggested that diminished sympathetic activity in brown adipose tissue may be a major cause of the reduced tissue thermogenic activity during lactation.
activity in the tissue by measuring the rate of noradrenaline turnover in brown adipose tissue from mid-lactating rats.
INTRODUCTION Brown adipose tissue shivering thermogenesis Frydman, 1978) and
is the major site for nonresponse to cold (Foster and overfeeding (Rothwell and
Stock, 1979) in rodents. The mechanism responsible for thermogenesis in brown fat is considered to be the mitochondrial proton conductance pathway described by Nicholls (1974). Thus, chronically cold+xposed rats show a high activity of the proton conductance pathway as well as an increase in the tissue size and mitochondrial content (Sundin and Cannon, 1980), and similar responses are observed in the tissue from chronically overfed rodents (Himms-Hagen et al., 1981). Brown fat thermogenesis has been reported to be suppressed in mid-lactating mice (Trayhurn et al., 1982) and rats (Villarroya et al., 1986) despite the hyperphagia that characterizes this physiological period. It has been suggested that diminished brown adipose tissue thermogenic activity during lactation would be an energy-sparing adaptation advantageous to the dam because of the nutritional stress linked to milk production. However, little is known about the regulatory mechanisms involved in this reduction of brown adipose tissue function. The sympathetic nervous system seems to play a key role in the regulation of brown adipose tissue activity. Thus, the sympathetic activity in brown fat-assessed from the rate of noradrenaline turnover-is increased in cold-exposed as well as in chronically-overfed rats (Young et al., 1982) and most of the brown adipose tissue modifications due to chronic cold exposure or overfeeding can be mimicked by noradrenaline treatment (Mory et al., 1984). The present work was undertaken to determine whether the reduced brown fat thermogenesis during lactation is related to changes in the sympathetic
Correspondence should be sent to Dr. Teresa Fisiologia Barcelona,
Mampel, General, Facultat de Biologia, Universitat de Avda Diagonal 645,08071 Barcelona, Spain. 481
MATERIALS AND METHODS Female Wistar rats (Rattus norvegicus) fed ad libitum Purina chow pellets (A04 type, Panlab, Barcelona) were mated with adult males when weighing 210-220 g. They were caged individually and maintained in a temperaturecontrolled environment (21 f 1°C). After parturition all litters were adjusted to 10 pups. Dams were used 15 days after parturition and compared with age-matched virgin controls. After decapitation of virgin and lactating rats the interscapular brown adipose tissue was removed, weighed and a small sample used for total lipid determination (Folch et al., 1957). Mitochondria were isolated from the remaining tissue (Cannon and Lindberg, 1979) and incubated for IO min with 10 PM (jH)-GDP essentially as described by Nicholls (1974) for the GDP-binding assay. Protein content of the mitochondrial preparations and the original tissue homogenates were determined (Wang and Smith, 1975) as well as the cytochrome oxidase activity (EC 1.9.3.1.) that was measured polarographically (Rafael, 1983). The recovery of cytochrome oxidase activity did not differ significantly between the two groups and the individual values were used to estimate the amount of mitochondrial protein in the interscapular brown adipose tissue and, subsequently, the total mitochondrial GDP-binding in the tissue. Statistical comparisons of the properties in brown fat from lactating and virgin control rats were performed with the Student’s t-test. Noradrenaline turnover was estimated in another set of 15-day lactating dams and virgin controls by measuring the noradrenaline concentration in interscapular brown adipose tissue at 0, 3 and 6 hr after i.p. injection of 80 mg/kg of a-methyl-p-tyrosine (Sigma, USA) as previously described (Spector et al., 1965; Young and Landsberg, 1977). After decapitation of the animals, interscapular brown adipose tissue was rapidly removed, weighed, homogenized in chilled 0.1 N perchloric acid containing. 0.1 mM reduced glutathione, and centrifuged at 0°C. Aliiuots of the supernatants were analyzed for noradrenaline content with a high performance liquid chromatography method (Eriksson and Persson, 1982) using a ~Bondapak C-18 column (Waters). The mobile phase was 0.04 M citrate/O. 1 M acetate buffer (PH 5.2) containing 10% methanol and 5 mM octanosulfonic acid. A flow rate of 1.5 ml/min was used and
482
FRANCESC VILLARROYA et al.
noradrenaline content was quantified with an electrochemical detector. Noradrenaline concentration data were set in a semi-
2000 c
logarithmic plot against time and the slope (fractional turnover rate, k) of the graph was calculated by the least squares method. Comparison of the fractional turnover rates was made using analysis of covariance. Noradrenaline turnover rates were calculated as the product of the fractional turnover rate and the endogenous noradrenaline concentration at the zero point. Ninety-five confidence intervals were determined for the noradrenaline turnover rates as described (Taubin et al., 1972).
RESULTS AND DISCUSSION
Composition, cytochrome oxidase activity and GDP-binding of interscapular brown adipose tissue from 15-day lactating rats compared with virgin controls are presented in Table 1. Interscapular brown fat weight was reduced in lactating rats to 69% of that of age-matched virgin controls. Tissue lipid content was not significantly altered by lactation whereas the protein content of interscapular brown adipose tissue from lactating rats was reduced. Both cytochrome oxidase specific activity, as well as whole tissue activity, were lower in lactating rats than in controls. Specific mitochondrial GDP-binding (nmol GDP/mg mitochondrial protein) was also significantly lower in dams and total mitochondrial GDP-binding (nmol GDP/interscapular brown fat) was reduced to 20% of that of virgin controls. These results indicate a generalized decrease in the activity of brown adipose tissue during lactation, essentially in agreement with previous reports in mid-lactating rats (Isler et al., 1984; Villarroya et al., 1986) and mice (Trayhurn et al., 1982). This low brown fat activity is due to the hypotrophy of the tissue, a decrease in its overall oxidative capacity-as shown by the cytochrome oxidase data-and a reduction in the mitochondrial proton conductance pathway activity estimated by the GDP-binding method. Data on noradrenaline content and turnover in interscapular brown adipose tissue from 15-day lactating rats are shown in Table 2. The slopes of noradrenaline concentration decay graphs are presented in Fig. 1. Noradrenaline concentrations
0
6
3
TIME
(hours)
Fig. 1. Noradrenaline turnover in interscapular brown adipose tissue from virgin and 1S-day lactating rats. All data are plotted as mean + SEM for endogenous noradrenaline in interscapular brown adipose tissues from 5 animals in each group at 0, 3 and 6 hr after the injection of a-methyl-p-tyrosine (80 mg/kg, i.p.). Closed circles represent virgin controls and open circles 15day lactating rats.
Virgin
Lactating
476.0 f 32.1
~- 330.1 + 28.lt
Lipid content (mg) per g of tissue per tissue
420.3 f 30.4 202.1 i 19.2
561.1 + 15.6: 185.0 f 18.2
were higher in interscapular brown adipose tissue from lactating dams but the total tissue noradrenaline content was not significantly changed. The fractional noradrenaline turnover rate (k) in the tissue was significantly reduced in lactating rats (an average 33% of that of virgin controls) while noradrenaline turnover was also significantly slower in the interscapular brown adipose tissue from dams (a mean 43% of that of virgin controls). The finding that noradrenaline turnover is reduced in interscapular brown fat from mid-lactating rats indicates that sympathetic activity in the tissue is reduced in this situation. Noradrenaline has been described to have a key role in the regulation of brown adipose tissue composition and activity (Desautels and Himms-Hagen, 1979; Mory et al., 1984) and our results suggest that low sympathetic nervous system activity in the tissue may have a major responsibility in the changes in brown fat described below leading to its lowered activity during lactation. Sympathetic activity in brown adipose tissue is considered to be closely related to food consumption (Landsberg and Young, 1983). Thus, fasted rats show reduced brown fat thermogenesis (Rothwell et al., 1984) associated with diminished sympathetic activity in the tissue (Young et al., 1982) whereas opposite changes take place in brown adipose tissue from overfed rats (Young et al., 1982). However, reduced brown adipose tissue thermogenesis and nor-
Protein content (mg) per g of tissue per tissue
144.0 + 6.1 67.5 +_ 3.9
122.1 f6.4’ 40.1 f 2.9:
Table 2. Noradrenaline content and turnover in interscapular brown adipose tissue from virgin and l5-day lactating rats
Table
Weight
I. Properties
of interscapular brown adipose tissue from virgin and I S-day lactating rats
(mg)
Cytochrome
Virgin
oxidase activity
(ymol O,/min) per mg of tissue protein per tissue GDP-binding (nmol GDP) per mg of mitochondrial protein per tissue
3.9 t 0.2 261.3 + 21.4
0.38 f 0.03 8.01 k 0.79
2.9 f 0.2t 119.7 * IO.71
0. I6 f 0.031 1.60fO.l3$
Results are means f SEM of 68 different animals. Statistical significance of the comparisons between virgin and lactating rats: ‘P 5 0.05; tP < 0.01; IP 5 0.001.
Noradrenaline (rig/g tissue) Noradrenaline (ngitissue) Noradrenaline fractional turnover rate, k (%/hr) Noradrenaline turnover (rig/g tissue/hr)
Lactating
1345 _t II2 646 + 90
1714 f 24* 512 & 45
5.1 + 0.5
I .8 + 0.6t
69. I f 5.7
30.0 I8.4t
Results are means + SEM of 5 animals for noradrenaline content data as well as for each time used to obtain the turnover data. Statistical significance of the comparisons between virgin and lactating rats: lP 5 0.05, tP 5 0.01.
Reduced NA turnover in BAT of lactating rats
adrenaline turnover in the tissue are associated with increased food consumption in pathological states such as the genetically obese (ob/ob) mice (Knehans and Romsos, 1982). Our results indicate that a reduction in the sympathetic activity in brown fat may also be the m~hanism responsible for the reduction in brown adipose tissue activity during lactation despite the associated hyperphagia. The mechanism for decreased thermogenesis in pathological states seems thus to be the same as in our physiological situation, suggesting a unity in the mechanisms responsible for brown fat modifications. On the other hand, even though the peripheral signals modulating sympathetic activity in relation to food intake are still not well known, circulating glucose and insulin have been claimed to be involved in this role (Landsberg and Young, 1983). In this sense, the low glucose and insulin levels occurring in the mid-lactating rat (Flint ef al., 1979; Burn01 et al., 1983) would be consistent with the reduced sympathetic activity found in brown fat in this situation. In summary, present results indicate that sympathetic activity, assessed by means of noradrenaline turnover, is reduced in brown adipose tissue from mid-lactating rats, in good correlation with a lower oxidative and thermogenic activity of the tissue in this situation. This finding suggests that low sympathetic nervous system activity in brown fat may be a major factor leading to its reduced thermogenic activity during lactation. Acknowledgemenr-The authors wish to express their gratitude to Maria Jose Sarrias from the Laboratory of Neurochemistry (C.S.I.C., Barcelona) for her help in the analysis of noradrenaline.
REFERENCES Burn01 A. F., Leturque A., Ferre P. and Girard J. (1983) Glucose metabolism during lactation in the rat: quantitative and regulatory aspects. Am. J. Physioi. 245, E351-E358. Cannon B. and Lindberg 0. (1979) Mitochondria from brown adipose tissue: isolation and properties. In Merhads in Enzymology (Edited by Fleischer S. and Packer L.) pp. 65-79. Academic Press, New York. Desautels M. and Himms-Hagen J. (1979) Roles of noradrenaline and protein synthesis in the cold-induced increase in purine nucleotide binding by rat brown adioose tissue mitochondria. Can. J. Phvsiol. 57.968-976. Eriksion B. M. and Persson B. A. (1982)Determination of catecholamines in rat heart tissue and plasma samples by liquid chromatography with electrochemical detector. J. Chromarogr. 228, 143- 154. Flint D. J., Sinnett-Smith P. A., Clegg R. A. and Vernon R. 6. (1979) Role of insulin receptors in the changing metabolism of adipose tissue during pregnancy and lactation in the rat. &hem. J. 182, 421-4>7. Folch J., Lees M. and Sloane-Stanley G. H. (1957) A simple method for the isolation and purification of the total lipids from animal tissues. J. biol. Chem. 226, 497-509.
483
Foster D. 0. and Frydman M. L. (1978) Non-shivering thermogenesis in the rat. II. Measurements of blood flow with microspheres point to brown adipose tissue as the dominant site of calorigenesis induced by noradrenaline. Can .J. Physiol. Pharmac. 56, 110-122.
Himms-Hagen J., Triand~llou J. and Gwiiliam C. (1981) Brown adipose tissue of cafeteria-fed rats. Am. J. Phy.~i~~/. 241, El 16E120. lsler D., Trayhurn P. and Lunn P. G. (I 984) Brown adipose tissue metabolism in lactating rats: the effect of litter size. Ann. Nutr. Metab. 28, 101-109.
Knehans A. W. and Romsos D. R. (1982) Reduced norepinephrine turnover in brown adipose tissue of the ob,/ob mice. Am. J. Ph.tziui. 242, E2533E261. Landsberg L. and Young J. B. (1983) Autonomic regulation of thermogenesis. in Mammalian Thermogenesis (Edited by Girardier L. and Stock M. J.) pp. 999140. Chapman and Hall, London. Mory G., Bouillaud F., Combes-George M. and Ricquier D. (1984) Noradrenaline controls the concentration of the uncoupling protein in brown adipose tissue. FEBS Lrtt. 166, 393-396. Nicholls D. G. (1974) Hamster brown adipose tissue mitochondria. The control of respiration and the proton electrochemical potential gradient by possible physiological effecters of the proton conductance of the inner membrane. Eur. J. Biochem. 37, 523 530. Rafael J, (1983) Cytochrome ox&se. In Methods q/ Enzymafic Analysi.s. (Edited by Bergmeyer H. V.), Vol 3. pp. 266283. Verlag Chimie, Base]. Rothwell N. J. and Stock M. J. (1979) A role for brown adipose tissue in diet-induced thermogenesis. Nururr (Lo&.) 281, 31-35. Rothwell N. J., Saville E. and Stock M. J. (1984) Brown fat activity in fasted and re-fed rats, Biosci. Rep. 4, 35 I 357. Spector S., Sjoerdsma A. and Udenfriend S. (1965) Blockade of endogenous norepinephrine synthesis by atphamethvhyrosine, an inhibitor of tyrosine hydroxylase. J. Pharmac. exp. Ther. 141, 8695.
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