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Brown fat, thermogenesis and physiological birth in a marsupial
Camp. Biorhcn~.Plrv.~io/Vol. 8lA. No. 4. pp. 815-819. 1985
0300-9629:85 $3.00+ 0.00
, 1985 Pergamon Pres\ Ltd
Printed in Great B;ita,n
BROWN
FAT,
THERMOGENESIS AND PHYSIOLOGICAL BIRTH IN A MARSUPIAL
ANSDKEWLOUDON*,
NANCY ROTHWELLt
and MICHAEL STocKt
*MRC/AFRC Comparative Physiology Research Group, Institute of Zoology, Regent’s Park, London, NWI 4RY and TDepartment of Physiology, St George’s Hospital Medical School, Tooting. London SW17 ORE, UK. Telephone: (01)672-1255 (Receiaed 26 October 1984) in young marsupials Abstract-l. Resting oxygen consumption corrected for body size, increased (Bennett’s Wallaby) with body weights of 5&4OOg. 2. Up to 250 g body wt injections of noradrenaline caused either a fall or no change in metabolic rate, but above this age a significant rise was evoked. 3. This age also corresponded to a rapid increase in growth rate and identification of active brown adiDose tissue. assessed from electron microscopy and measurements of purine nucleotide binding to isolated mitochondria.
INTRODUCTION
(Dawson and Hulbert, 1970), but these animals are capable of efficient thermoregulation (Shield, 1966; Wallis and Mayers, 1973; Setchell, 1974). Thus, at some stage, the marsupial develops thermoregulatory mechanisms that allow it to change from an essentially ectothermic to an endothermic animal. Since non-shivering thermogenesis is most apparent in neonatal eutherians, the relatively under-developed state of the newborn marsupial provides a unique opportunity for the study of the ontogeny of mammalian thermoregulatory mechanisms in an easily available exteriorized fetus. In this study, thermogenesis in the pouched-young of the wallaby was investigated in an attempt to elucidate both the mechanisms and the development of thermoregulation in a marsupial over a range of developmental states, equivalent to the eutherian fetal and neonatal period of life.
Sympathetic activation of brown adipose tissue (BAT) heat production is now widely recognized as the principal source of thermoregulatory, nonshivering thermogenesis in hibernators, neonates and cold-adapted eutherians (Foster and Frydman, 1978; Girardier, 1983). The very high thermogenic capacity of brown fat has been ascribed to the presence of a unique proton conductance pathway within the inner mitochondrial membrane, which allows uncoupled respiration (Nicholls, 1983). The activity of this pathway can be assessed from the binding of purine nucleotides to isolated mitochondria, which again seems to be unique to BAT. We have recently identified two distinct purine nucleotide binding sites from Scatchard analysis and the number of high affinity binding sites is sensitive to acute thermogenic stimuli (Bryant et ul., 1983). Although these biochemical measurements can be used to assess the specific activity of BAT, an estimate of the total thermogenic capacity of an animal can most easily be obtained from the rise in metabolic rate following infusion or injection of noradrenaline. This is largely due to an increase in oxygen consumption of brown adipose tissue, at least in laboratory rodents (Foster and Frydman, 1978; Rothwell and Stock, 1981). Although BAT has been found in many orders of mammals, studies to date have failed to identify the tissue in marsupials (metatherians) and it has been suggested that this subclass lacks brown fat (Rowlatt er al., 1971). However, adult marsupials are capable of regulating body temperature in response to cold, although the mechanisms involved have not been well described (Smith and Dawson, 1984). Marsupials are characterized by very short gestations (generally < 30 days) and long lactations, so that most of their development must occur post-natally. Young marsupial joeys show very low basal metabolic rates and are apparently unable to activate heat production in response to cold (Shield, 1966; Wallis and Mayers, 1973). Adult metabolic rates for marsupials generally remain at about 25-3O”A below eutherian levels
MATERIALS AND METHODS
Six adult lactating Bennett’s wallabies (Macropus rz@ griseus rufogriseus) were maintained as a group on an ad lihitum diet of lucerne from September. through to July of the following year. Joeys were born in early October after a 28.day gestation and weighed 0.7 g (+ I g SEM) at birth. Measurements were made each week of the growth rate of the joey. Joeys of 30- I50 g were weighed still attached to the teat, with a small spring . - balance while the mothers were temporarily anaesthetized (ketamine 50 mg. xylazine 200 mg). From 150 y body wt onwards, ioevs were detached from the teat if necessar; and weighed-oitside the pouch. Previous work has established that there is a close correlation between age and pouch weight (Fleming el al., l983), and that such attachment has no effect on subsequent growth. Studies on metabolic rate were made on a total of 34 joeys (of between 5 and 500g body wt) removed permanently from females captured at the Bennett’s wallaby colony (Whipsnade Park, Bedford). All measurements commenced within an hour of removal from the pouch and joeys were maintained during this period in an incubator at 37 C and over 80% humidity. Animals were placed in black cloth bags and resting oxygen consumption ( Vo,) was measured in closed circuit respirometers (Stock, 1975) at 37‘C (1OO:/0 humidity) for 815
816
ANDREW
LOUDON
50-90 min before. and up to 90 min after. a single subcutaneous injection of noradrenahne (40 pg/lOO g body wt). No treatment with noradrenaline was made of joeys of less than 100 g body wt. All values for metabolic rate were corrected for metabolic body size (ml/min/kgo75). After these measurements. the animals were killed by decapitation and all visible internal adipose tissue dissected and weighed. Small samples were taken from some animals and chopped, fixed in 3”; gluteraldehyde and post-fixed in 1”; osmium tetroxide in 0.1 M cocodylate buffer (pH 7.4). Sections were then taken for electron microscopy and were stained with uranyl acetate and lead citrate. The remaining adipose tissue was minced, homogenized in 0.2 M sucrose and mitochondria were prepared (Slinde et al., 1983). The binding of [‘Hlguanosine diphosphate (GDP. Amersham International, Bucks. UK, 10 Ci/mmol) to isolated mitochondria was assessed as previously (Bryant rr crl., 1984) using a range of concentrations from 35 nM to 10 PM GDP and 14C sucrose to estimate inter-mitochondrial space. Non-specific binding was assessed in the presence of 200 p M unlabelled nucleotide. Apparent dissociation constants (&) and maximum number of binding sites (B,,,) were calculated from Scatchard analysis of binding data, which were resolved into one or two lines by interactive computer analysis, as described previously (Bryant et al., 1983. 1984). Mitochondrial and total protein contents were assessed by a dye reagent method (Bio-Rad. Watford, UK). Values are presented as means + SEM.
et al
Table
I. Specific mitochondrial GDP-binding tissue from young joeys site
wt range (Es)
I
capacity
of adipose
Site 11
B m*\ & (JIM) (pmol/mgP)
B ml”
4
(PM)
125-145
(pmol/mgP)
I
Error+
20
0.5
0.77
39
0.4
0.2
(3) 135Sl80
~
(3) 230.-255
0.09
4
0.76
32
0 3
0.04
II
5.00
52
II.3
0.03
4
1.52
36
06
0.20
IO
0 7
2.00
50
0.4
(3) 35&460 (3) 57x (1) Rat WAT (6) Rat BAT
0.05
5
(6) (n) = number of animals from which tissue was pooled. tError presented as (I - r)(;“. Data from white and brown adipose tissue from young adult rats (rat WAT. rat BAT) were obtained in separate studies.
Adult marsupial mterspeces
rate
RESULTS
Resting Vo2corrected for body size (kg”‘) was very low in young joeys up to 110 days old (Fig. lA), compared to eutherian values (25-30 times lower) and increased with body wt from 10 to 400 g (i.e. 2&135 days old) by which time values were similar to those obtained for adult marsupials. A single injection of noradrenaline caused a significant (P < 0.05) reduction in metabolic rate in joeys weighing lOO_200g (approx. 85 days), no change in animals at 20&300 g, and a significant (P < 0.001) rise (48 + 6%) in joeys weighing 350-580 g (I 35 days old, Fig. 1B). Positive responses to noradrenaline were seen only in animals whose eyes had opened and the time course of this response (peak at 30-40 min) was identical to the reduction in oxygen consumption seen in younger joeys. Very little adipose tissue of any description (l-3 g) was seen in any of the joeys and the fat which was found in the cervical, interscapular and axillary regions was pooled from several animals. Tissue in these areas appeared white in colour in younger animals (< 250 g) and electron microscopy revealed the typical appearance of white adipose tissue (large unilocular fat droplets, sparse mitochondria). In older joeys, the tissue appeared darker in colour and under the electron microscope, cells typical of those found in eutherian brown fat were seen (numerous, densely-packed mitochondria, small multilocular fat droplets and many blood vessels) as shown in Fig. 2. Scatchard analysis of ‘H-GDP binding to mitochondria isolated from young joeys (>200 g) revealed linear plots, indicating a single class of binding sites (Table 1). The Kd and B,,, of this site were similar to those found for rat white adipose tissue in separate studies (Table 1). In joeys above 200 g body wt, curvilinear Scatchards were obtained and these were resolved into two lines (Bryant et al., 1983),
B
i------y , __ .__ ._
100
200 Body
300 weight
400
500
I gl
Fig. I. (A) Resting oxygen consumption (Vo,) of young male and female wallabies (5-580 g live weight), corrected for metabolic body size (mlO,/min/kg” “). Values represent means of 3-8 animals + SEM. The adult marsupial interspecies rate is (Dawson and Hulbert, 1970) shown for comparative purposes. (B) Percentage increase in resting Yo, in joeys of different ages following a single intra-peritoneal injection of noradrenaline (40 pg/lOO g live weight). Resting k’o, was measured for SO-90 min before and after injection. Each value represents a mean of 335 animals ( f SEM). (C) Growth rates of young joeys (g live weight gain/day) for six captive lactating wallabies. The letters A-E refer to: (A) occasional voluntary teat detachment (approx. 70 days old); (B) eyes open (100 days); (C) hair growth; (D) detached 50’:” of time (125 days); (E) head out of pouch (140 days).
817
Wallaby brown fat
Fig. 2. Electron
micrograph
of brown adipose tissue taken from the interscapular Bar = 5 pm (Magnification x 9OOq).
since the errors associated with a two site plot were at least 10 times lower than resolution of the points into a single line. The dissociation constants and maximum number of binding sites for both of these sites on joey mitochondria, were quantitatively similar to those obtained on active brown fat from the rat (see Table 1). DISCUSSION
Growth of marsupials is characterized by an initial slow growth rate (i.e. over the first 5 months in the Bennett’s wallaby), followed by an acceleration in later life and this can be seen in Fig. lC, where an increase in growth occurs after first teat detachment at about 90 days old. These two phases have been considered to correspond quantitatively and qualitatively with fetal and postnatal development in eutherians (Russell, 1982), the point when growth increases being suggested by some to be the physiological equivalent of birth in a eutherian (Mayers, 1976). The rise in growth rate of Bennett’s wallabies correlated with an increase in metabolic rate (even when corrected for metabolic body size, Fig. 1A) and the appearance of positive thermogenic responses to noradrenaline (Fig. 1B). The oxygen consumption of very young joeys (< 100 g) is extremely low, presumably largely because of their poikilothermic state, but by 500 g body wt, resting rates of energy expenditure in these animals were similar to those quoted for adult marsupials (Dawson and Hulbert, 1970). At this stage joeys spend a large proportion of the day with their head and fore limbs out of the pouch, short-time pouch exit (< 5 rain) can occur at 600-700 g (145 days) and joeys leave the pouch for
region of a 400 g joey.
longer periods by 1.2-l .5 kg (190 days). Permanent pouch exit occurs at about 2.5 kg (225 days), although joeys are not completely weaned until they reach a weight of 6-7 kg, some three months later. The reasons for the reduction in oxygen consumption of younger joeys after injections of noradrenaline (Fig. 1B) is not known, but could be related to a redistribution of tissue blood flow. However, a positive response to noradrenaline was obtained in all joeys over 230 g body wt and the magnitude of the response in the oldest joeys (400 g-500 g) was comparable to that seen in rodents (Rothwell and Stock, 1979). Thermogenic responses to noradrenaline are used as a standard test of the capacity for non-shivering thermogenesis in mammals and are largely due to activation of heat production in BAT, at least in rodents and lagomorphs (see Foster and Frydman, 1978; Girardier, 1983; Rothwell and Stock, 1984 for reviews). Very little adipose tissue was seen in the joeys in this study and in the younger animals this appeared, from histological and biochemical measurements, to correspond to white adipose tissue, although the distribution was similar to that normally described for BAT (i.e. axillary, cervical). However, in older joeys electron microscopy revealed structural features typical of those found in eutherian brown fat. The principal thermogenic pathway in BAT appears to be a unique mitochondrial proton conductance pathway, the activity of which can be assessed from mitochondrial purine nucleotide binding (Nicholls and Locke, 1983). Scatchard analysis of 3H-guanosine binding to mitochondria isolated from wallaby adipose tissue revealed a low number of a single class of binding sites in young animals
818
ANDREW
LOUDON et al.
(< 230 g, Table l), similar to that seen in rat white adipose tissue. However, in older animals two distinct binding sites were observed, and by 35&500 g body wt the apparent dissociation constants (&) and maximum capacities (B,,,) of both sites were similar to those observed in rodents with active BAT (Bryant et al., 1983). This suggests that the specific activity of the mitochondrial proton conductance pathway increased with age and the appearance of the two distinct GDP-binding sites corresponded to the time when thermogenic responses to noradrenaline could be elicited and resting metabolic rates had risen to levels similar to those quoted for adult marsupials. A high density of /?-adrenergic receptors on isolated adipose tissue membranes was identified using Scatchard analysis of ( -)-3[H]dihydroalprenolol binding (Loudon et al., unpublished data). The density of these receptors did not vary with age (13&180 g joeys; B,,, = 254 fmol/mg protein, K,, = 2.93 nM; 35tX4.50 g joeys; B,,, = 257 fmol/mg protein, Kd = 2.70 nM) and these values were similar to those obtained for brown and white adipose tissue membranes in the rat (e.g. rat BAT: B,,, = 160 fmol/ mg protein, Kd = 2 nM). Thus, the inability of young joeys to raise heat production in response to noradrenaline is presumably due to an absence of the mitochondrial proton conductance pathway. rather than a failure of catecholamines to interact with the cell membrane. One important modulator of b-adrenoreceptor density, which can also exert profound effects on metabolic rate, is thyroid hormone. However, circulating levels of triiodothyronine (T,) were similar in joeys of all ages studied (440 + 50 pg/ml). This infers that the marked changes in growth, metabolic rate and responses to noradrenaline were not due to changes in circulating T, levels, although changes in thyroid hormone turnover or interactions with nuclear receptors may have occurred. A further unusual feature of brown fat is that it contains high levels of phosphocreatine kinase (PCK) which are comparable to that of muscle (rat brown fat = 5-6, white fat = I, muscle = 4 units/mg protein; T. Peters and A. Poustie, personal communication). Levels of PCK found by these workers in adipose tissue from joeys weighing over 250 g were comparable (47 units/mg protein), to those found in rat brown fat. Thus, by criteria accepted for eutherians, the young joey appears to possess active brown fat. In very young animals (~200 g) adipose tissue was observed in areas where brown fat is found in eutherians and where it was subsequently observed in the older joeys (i.e. interscapular and axillary regions). However, it is not known whether thermogenie brown adipocytes develop in the interscapular and axillary regions from a separate cell line, or whether they differentiate from the white adipocytes already present in the young joey. The problem of the ontogeny of white and brown fat cells is still largely unresolved for eutherians, mainly because of the difficulties of studies in utero; marsupials, with their exteriorized fetus, may provide an easier system for further studies in this area. It seems that the development of brown adipose tissue and thermogenic responses to noradrenaline precedes, by approx. one month, the time when joeys
leave the pouch and in their natural environment (Tasmania), are forced to thermoregulate. The appearance of these thermoregulatory characteristics at this stage of development corresponds to the appearance of the same metabolic features in perinatal eutherians. In this respect, marsupials are no different from eutherians, except that marsupial parturition is followed by an extension of fetal life as a prelude to physiological birth. The existence and importance of BAT has been considered to be limited to only a feu orders of eutherian mammals, but the present study, together with the recent histological identification of this tissue in birds (Oliphant, 1983) and the report of a homologous “brain heater” in the swordfish (Carey, 1982) suggests that this unique heatproducing tissue has a far wider biological significance within the animal kingdom. Acknowledgemenfs~We wish to thank Tim Peters and Alison Poustie (MRC Clinical Research Centre) for measuring phosphocreatine kinase activity in samples of adipose tissue and Richard Cinderey (Whipsnade Park) for assistance with the animals. This work was supported by a grant from ICI (Joint Research Scheme), a programme grant from the MRC and AFRC to Prof. J. Hearn and by a grant for studies in mammalian growth from Marks and Spencer. PLC. NJR is a Royal Society Research Fellow. REFERENCES Bryant K. R., Rothwell N. J. and Stock M. J. (1983) Identification of two mitochondrial GDP-binding sites in rat brown adipose tissue. Biosci. Rep. 3, 589-598. Bryant K. R., Rothwell N. J. and Stock M. J. (1984) Acute influences in the two GDP binding sites in brown adipose tissue mitochondria. Biosci. Rep. 4, 5233533. Carey F. G. (I 982) A brain heater in the sword fish. Science 216, 1327-1329. Dawson T. J. and Hulbert A. J. (1970) Standard metabolism, body temperature, and surface areas of Australian marsupials. Am. J. Ph.rsiol. 218, 123331238. Fleming D.. Cinderev R. W. and Hedrn J. P. (1983) The reproductive biology of the Bennett’s wallaby (Macropus r~fogriseus rufogri.seu,s) living fret at Whipsnade Park. J. Zool., Lo&. 201, 283329 I. Foster D. 0. and Frydman M. L. (1978) Nonshivering thermogenesis in the rat. II. Measurements of blood flow with microspheres point to brown adipose tissue as the dominant site of the calorigenesis induced by noradrenaline. Can. J. Physioi. Pharmacol. 56, 110~122. Girardier L. (1983) Brown fat: An energy dissipating tissue. In Mammalian Thermogenesis (Edited by Girardier L. and Stock M. J.), pp. 50-98. Chapman & Hall, London. Mavnes G. (1976)Growth of the parma wallaby, Macropus phrma Waterhouse. Austr. J. .&I. 24, 217-236. Morrison P. and Petajan J. H. (1962) The development of temperature regulation in the opossum, (Didelphis marsupialis oirginiana). Phq’siol. Zool. 35, 52-65. Nicholls D. G. and Locke R. (I 983) Cellular mechanisms of heat dissipation. In Mammalian Thermogenesis (Edited by Girardier L. and Stock M. J.). pp. 849. Chapman & Hall, London. Oliphant R. L. W. (1983) First observations of brown fat m birds. Condor 85, 350-354. Rothwell N. J. and Stock M. J. (1979) A role for brown adipose tissue in diet-induced thermogenesis. Nature 281, 31-35. Rothwell N. J. and Stock M. J. (1984) Brown adipose tissue. In Recent Adwnces in Pll,sio/ogy (Edited by Baker P. F.). Vol. 10. pp. 208233. Churchill Livingstone, Edinburgh.
Wallaby Rowlatt U., Mrosovsky N. and English A. (1971) A comparative survey of brown fat in the neck and axilla of mammals at birth. Biol. Neonate 17, 53-84. Russell E. M. (1982) Patterns of parental care and parental investment in marsupials. Biol. Rev. 57, 423486. Shield J. (1966) Oxygen consumption during pouch development of the macropod marsupial. Setonix brachyurus (quokka). J. Physiol. 187, 257-270. Setchell P. J. (1974) The development of thermoregulation and thyroid function in the marsupial Macropus eujenii (Desmarest). Camp. Biochem. Physiol. 47A, 1115-l 121. Slinde E., Pederson J. J. and Flatmark T. (1975) Sedimentation coefficient and buoyant density of brown adipose
brown
fat
819
tissue mitochondria from guinea pigs. Analyr. Biochem. 65, 581-585. Smith B. K. and Dawson T. J. (1984) Effects of cold and warm acclimation on the thermal balance of a marsupial (Dasyuroides bymei). In Thermal Physiology (Edited by Hales J. R. S.), pp. 475478. Raven Press, New York. Stock M. J. (1975) An automatic, closed-circuit oxygen consumption apparatus for small animals. J. appl. Physiol. 39, 849-850. Wallis R. L. and Maynes G. M. (1973) Ontogeny of thermoregulation in Macropus parma (Marsupialia macropodidae). J. Mammal. 54, 278-28 1.
0300-9629:85 $3.00+ 0.00
, 1985 Pergamon Pres\ Ltd
Printed in Great B;ita,n
BROWN
FAT,
THERMOGENESIS AND PHYSIOLOGICAL BIRTH IN A MARSUPIAL
ANSDKEWLOUDON*,
NANCY ROTHWELLt
and MICHAEL STocKt
*MRC/AFRC Comparative Physiology Research Group, Institute of Zoology, Regent’s Park, London, NWI 4RY and TDepartment of Physiology, St George’s Hospital Medical School, Tooting. London SW17 ORE, UK. Telephone: (01)672-1255 (Receiaed 26 October 1984) in young marsupials Abstract-l. Resting oxygen consumption corrected for body size, increased (Bennett’s Wallaby) with body weights of 5&4OOg. 2. Up to 250 g body wt injections of noradrenaline caused either a fall or no change in metabolic rate, but above this age a significant rise was evoked. 3. This age also corresponded to a rapid increase in growth rate and identification of active brown adiDose tissue. assessed from electron microscopy and measurements of purine nucleotide binding to isolated mitochondria.
INTRODUCTION
(Dawson and Hulbert, 1970), but these animals are capable of efficient thermoregulation (Shield, 1966; Wallis and Mayers, 1973; Setchell, 1974). Thus, at some stage, the marsupial develops thermoregulatory mechanisms that allow it to change from an essentially ectothermic to an endothermic animal. Since non-shivering thermogenesis is most apparent in neonatal eutherians, the relatively under-developed state of the newborn marsupial provides a unique opportunity for the study of the ontogeny of mammalian thermoregulatory mechanisms in an easily available exteriorized fetus. In this study, thermogenesis in the pouched-young of the wallaby was investigated in an attempt to elucidate both the mechanisms and the development of thermoregulation in a marsupial over a range of developmental states, equivalent to the eutherian fetal and neonatal period of life.
Sympathetic activation of brown adipose tissue (BAT) heat production is now widely recognized as the principal source of thermoregulatory, nonshivering thermogenesis in hibernators, neonates and cold-adapted eutherians (Foster and Frydman, 1978; Girardier, 1983). The very high thermogenic capacity of brown fat has been ascribed to the presence of a unique proton conductance pathway within the inner mitochondrial membrane, which allows uncoupled respiration (Nicholls, 1983). The activity of this pathway can be assessed from the binding of purine nucleotides to isolated mitochondria, which again seems to be unique to BAT. We have recently identified two distinct purine nucleotide binding sites from Scatchard analysis and the number of high affinity binding sites is sensitive to acute thermogenic stimuli (Bryant et ul., 1983). Although these biochemical measurements can be used to assess the specific activity of BAT, an estimate of the total thermogenic capacity of an animal can most easily be obtained from the rise in metabolic rate following infusion or injection of noradrenaline. This is largely due to an increase in oxygen consumption of brown adipose tissue, at least in laboratory rodents (Foster and Frydman, 1978; Rothwell and Stock, 1981). Although BAT has been found in many orders of mammals, studies to date have failed to identify the tissue in marsupials (metatherians) and it has been suggested that this subclass lacks brown fat (Rowlatt er al., 1971). However, adult marsupials are capable of regulating body temperature in response to cold, although the mechanisms involved have not been well described (Smith and Dawson, 1984). Marsupials are characterized by very short gestations (generally < 30 days) and long lactations, so that most of their development must occur post-natally. Young marsupial joeys show very low basal metabolic rates and are apparently unable to activate heat production in response to cold (Shield, 1966; Wallis and Mayers, 1973). Adult metabolic rates for marsupials generally remain at about 25-3O”A below eutherian levels
MATERIALS AND METHODS
Six adult lactating Bennett’s wallabies (Macropus rz@ griseus rufogriseus) were maintained as a group on an ad lihitum diet of lucerne from September. through to July of the following year. Joeys were born in early October after a 28.day gestation and weighed 0.7 g (+ I g SEM) at birth. Measurements were made each week of the growth rate of the joey. Joeys of 30- I50 g were weighed still attached to the teat, with a small spring . - balance while the mothers were temporarily anaesthetized (ketamine 50 mg. xylazine 200 mg). From 150 y body wt onwards, ioevs were detached from the teat if necessar; and weighed-oitside the pouch. Previous work has established that there is a close correlation between age and pouch weight (Fleming el al., l983), and that such attachment has no effect on subsequent growth. Studies on metabolic rate were made on a total of 34 joeys (of between 5 and 500g body wt) removed permanently from females captured at the Bennett’s wallaby colony (Whipsnade Park, Bedford). All measurements commenced within an hour of removal from the pouch and joeys were maintained during this period in an incubator at 37 C and over 80% humidity. Animals were placed in black cloth bags and resting oxygen consumption ( Vo,) was measured in closed circuit respirometers (Stock, 1975) at 37‘C (1OO:/0 humidity) for 815
816
ANDREW
LOUDON
50-90 min before. and up to 90 min after. a single subcutaneous injection of noradrenahne (40 pg/lOO g body wt). No treatment with noradrenaline was made of joeys of less than 100 g body wt. All values for metabolic rate were corrected for metabolic body size (ml/min/kgo75). After these measurements. the animals were killed by decapitation and all visible internal adipose tissue dissected and weighed. Small samples were taken from some animals and chopped, fixed in 3”; gluteraldehyde and post-fixed in 1”; osmium tetroxide in 0.1 M cocodylate buffer (pH 7.4). Sections were then taken for electron microscopy and were stained with uranyl acetate and lead citrate. The remaining adipose tissue was minced, homogenized in 0.2 M sucrose and mitochondria were prepared (Slinde et al., 1983). The binding of [‘Hlguanosine diphosphate (GDP. Amersham International, Bucks. UK, 10 Ci/mmol) to isolated mitochondria was assessed as previously (Bryant rr crl., 1984) using a range of concentrations from 35 nM to 10 PM GDP and 14C sucrose to estimate inter-mitochondrial space. Non-specific binding was assessed in the presence of 200 p M unlabelled nucleotide. Apparent dissociation constants (&) and maximum number of binding sites (B,,,) were calculated from Scatchard analysis of binding data, which were resolved into one or two lines by interactive computer analysis, as described previously (Bryant et al., 1983. 1984). Mitochondrial and total protein contents were assessed by a dye reagent method (Bio-Rad. Watford, UK). Values are presented as means + SEM.
et al
Table
I. Specific mitochondrial GDP-binding tissue from young joeys site
wt range (Es)
I
capacity
of adipose
Site 11
B m*\ & (JIM) (pmol/mgP)
B ml”
4
(PM)
125-145
(pmol/mgP)
I
Error+
20
0.5
0.77
39
0.4
0.2
(3) 135Sl80
~
(3) 230.-255
0.09
4
0.76
32
0 3
0.04
II
5.00
52
II.3
0.03
4
1.52
36
06
0.20
IO
0 7
2.00
50
0.4
(3) 35&460 (3) 57x (1) Rat WAT (6) Rat BAT
0.05
5
(6) (n) = number of animals from which tissue was pooled. tError presented as (I - r)(;“. Data from white and brown adipose tissue from young adult rats (rat WAT. rat BAT) were obtained in separate studies.
Adult marsupial mterspeces
rate
RESULTS
Resting Vo2corrected for body size (kg”‘) was very low in young joeys up to 110 days old (Fig. lA), compared to eutherian values (25-30 times lower) and increased with body wt from 10 to 400 g (i.e. 2&135 days old) by which time values were similar to those obtained for adult marsupials. A single injection of noradrenaline caused a significant (P < 0.05) reduction in metabolic rate in joeys weighing lOO_200g (approx. 85 days), no change in animals at 20&300 g, and a significant (P < 0.001) rise (48 + 6%) in joeys weighing 350-580 g (I 35 days old, Fig. 1B). Positive responses to noradrenaline were seen only in animals whose eyes had opened and the time course of this response (peak at 30-40 min) was identical to the reduction in oxygen consumption seen in younger joeys. Very little adipose tissue of any description (l-3 g) was seen in any of the joeys and the fat which was found in the cervical, interscapular and axillary regions was pooled from several animals. Tissue in these areas appeared white in colour in younger animals (< 250 g) and electron microscopy revealed the typical appearance of white adipose tissue (large unilocular fat droplets, sparse mitochondria). In older joeys, the tissue appeared darker in colour and under the electron microscope, cells typical of those found in eutherian brown fat were seen (numerous, densely-packed mitochondria, small multilocular fat droplets and many blood vessels) as shown in Fig. 2. Scatchard analysis of ‘H-GDP binding to mitochondria isolated from young joeys (>200 g) revealed linear plots, indicating a single class of binding sites (Table 1). The Kd and B,,, of this site were similar to those found for rat white adipose tissue in separate studies (Table 1). In joeys above 200 g body wt, curvilinear Scatchards were obtained and these were resolved into two lines (Bryant et al., 1983),
B
i------y , __ .__ ._
100
200 Body
300 weight
400
500
I gl
Fig. I. (A) Resting oxygen consumption (Vo,) of young male and female wallabies (5-580 g live weight), corrected for metabolic body size (mlO,/min/kg” “). Values represent means of 3-8 animals + SEM. The adult marsupial interspecies rate is (Dawson and Hulbert, 1970) shown for comparative purposes. (B) Percentage increase in resting Yo, in joeys of different ages following a single intra-peritoneal injection of noradrenaline (40 pg/lOO g live weight). Resting k’o, was measured for SO-90 min before and after injection. Each value represents a mean of 335 animals ( f SEM). (C) Growth rates of young joeys (g live weight gain/day) for six captive lactating wallabies. The letters A-E refer to: (A) occasional voluntary teat detachment (approx. 70 days old); (B) eyes open (100 days); (C) hair growth; (D) detached 50’:” of time (125 days); (E) head out of pouch (140 days).
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Wallaby brown fat
Fig. 2. Electron
micrograph
of brown adipose tissue taken from the interscapular Bar = 5 pm (Magnification x 9OOq).
since the errors associated with a two site plot were at least 10 times lower than resolution of the points into a single line. The dissociation constants and maximum number of binding sites for both of these sites on joey mitochondria, were quantitatively similar to those obtained on active brown fat from the rat (see Table 1). DISCUSSION
Growth of marsupials is characterized by an initial slow growth rate (i.e. over the first 5 months in the Bennett’s wallaby), followed by an acceleration in later life and this can be seen in Fig. lC, where an increase in growth occurs after first teat detachment at about 90 days old. These two phases have been considered to correspond quantitatively and qualitatively with fetal and postnatal development in eutherians (Russell, 1982), the point when growth increases being suggested by some to be the physiological equivalent of birth in a eutherian (Mayers, 1976). The rise in growth rate of Bennett’s wallabies correlated with an increase in metabolic rate (even when corrected for metabolic body size, Fig. 1A) and the appearance of positive thermogenic responses to noradrenaline (Fig. 1B). The oxygen consumption of very young joeys (< 100 g) is extremely low, presumably largely because of their poikilothermic state, but by 500 g body wt, resting rates of energy expenditure in these animals were similar to those quoted for adult marsupials (Dawson and Hulbert, 1970). At this stage joeys spend a large proportion of the day with their head and fore limbs out of the pouch, short-time pouch exit (< 5 rain) can occur at 600-700 g (145 days) and joeys leave the pouch for
region of a 400 g joey.
longer periods by 1.2-l .5 kg (190 days). Permanent pouch exit occurs at about 2.5 kg (225 days), although joeys are not completely weaned until they reach a weight of 6-7 kg, some three months later. The reasons for the reduction in oxygen consumption of younger joeys after injections of noradrenaline (Fig. 1B) is not known, but could be related to a redistribution of tissue blood flow. However, a positive response to noradrenaline was obtained in all joeys over 230 g body wt and the magnitude of the response in the oldest joeys (400 g-500 g) was comparable to that seen in rodents (Rothwell and Stock, 1979). Thermogenic responses to noradrenaline are used as a standard test of the capacity for non-shivering thermogenesis in mammals and are largely due to activation of heat production in BAT, at least in rodents and lagomorphs (see Foster and Frydman, 1978; Girardier, 1983; Rothwell and Stock, 1984 for reviews). Very little adipose tissue was seen in the joeys in this study and in the younger animals this appeared, from histological and biochemical measurements, to correspond to white adipose tissue, although the distribution was similar to that normally described for BAT (i.e. axillary, cervical). However, in older joeys electron microscopy revealed structural features typical of those found in eutherian brown fat. The principal thermogenic pathway in BAT appears to be a unique mitochondrial proton conductance pathway, the activity of which can be assessed from mitochondrial purine nucleotide binding (Nicholls and Locke, 1983). Scatchard analysis of 3H-guanosine binding to mitochondria isolated from wallaby adipose tissue revealed a low number of a single class of binding sites in young animals
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LOUDON et al.
(< 230 g, Table l), similar to that seen in rat white adipose tissue. However, in older animals two distinct binding sites were observed, and by 35&500 g body wt the apparent dissociation constants (&) and maximum capacities (B,,,) of both sites were similar to those observed in rodents with active BAT (Bryant et al., 1983). This suggests that the specific activity of the mitochondrial proton conductance pathway increased with age and the appearance of the two distinct GDP-binding sites corresponded to the time when thermogenic responses to noradrenaline could be elicited and resting metabolic rates had risen to levels similar to those quoted for adult marsupials. A high density of /?-adrenergic receptors on isolated adipose tissue membranes was identified using Scatchard analysis of ( -)-3[H]dihydroalprenolol binding (Loudon et al., unpublished data). The density of these receptors did not vary with age (13&180 g joeys; B,,, = 254 fmol/mg protein, K,, = 2.93 nM; 35tX4.50 g joeys; B,,, = 257 fmol/mg protein, Kd = 2.70 nM) and these values were similar to those obtained for brown and white adipose tissue membranes in the rat (e.g. rat BAT: B,,, = 160 fmol/ mg protein, Kd = 2 nM). Thus, the inability of young joeys to raise heat production in response to noradrenaline is presumably due to an absence of the mitochondrial proton conductance pathway. rather than a failure of catecholamines to interact with the cell membrane. One important modulator of b-adrenoreceptor density, which can also exert profound effects on metabolic rate, is thyroid hormone. However, circulating levels of triiodothyronine (T,) were similar in joeys of all ages studied (440 + 50 pg/ml). This infers that the marked changes in growth, metabolic rate and responses to noradrenaline were not due to changes in circulating T, levels, although changes in thyroid hormone turnover or interactions with nuclear receptors may have occurred. A further unusual feature of brown fat is that it contains high levels of phosphocreatine kinase (PCK) which are comparable to that of muscle (rat brown fat = 5-6, white fat = I, muscle = 4 units/mg protein; T. Peters and A. Poustie, personal communication). Levels of PCK found by these workers in adipose tissue from joeys weighing over 250 g were comparable (47 units/mg protein), to those found in rat brown fat. Thus, by criteria accepted for eutherians, the young joey appears to possess active brown fat. In very young animals (~200 g) adipose tissue was observed in areas where brown fat is found in eutherians and where it was subsequently observed in the older joeys (i.e. interscapular and axillary regions). However, it is not known whether thermogenie brown adipocytes develop in the interscapular and axillary regions from a separate cell line, or whether they differentiate from the white adipocytes already present in the young joey. The problem of the ontogeny of white and brown fat cells is still largely unresolved for eutherians, mainly because of the difficulties of studies in utero; marsupials, with their exteriorized fetus, may provide an easier system for further studies in this area. It seems that the development of brown adipose tissue and thermogenic responses to noradrenaline precedes, by approx. one month, the time when joeys
leave the pouch and in their natural environment (Tasmania), are forced to thermoregulate. The appearance of these thermoregulatory characteristics at this stage of development corresponds to the appearance of the same metabolic features in perinatal eutherians. In this respect, marsupials are no different from eutherians, except that marsupial parturition is followed by an extension of fetal life as a prelude to physiological birth. The existence and importance of BAT has been considered to be limited to only a feu orders of eutherian mammals, but the present study, together with the recent histological identification of this tissue in birds (Oliphant, 1983) and the report of a homologous “brain heater” in the swordfish (Carey, 1982) suggests that this unique heatproducing tissue has a far wider biological significance within the animal kingdom. Acknowledgemenfs~We wish to thank Tim Peters and Alison Poustie (MRC Clinical Research Centre) for measuring phosphocreatine kinase activity in samples of adipose tissue and Richard Cinderey (Whipsnade Park) for assistance with the animals. This work was supported by a grant from ICI (Joint Research Scheme), a programme grant from the MRC and AFRC to Prof. J. Hearn and by a grant for studies in mammalian growth from Marks and Spencer. PLC. NJR is a Royal Society Research Fellow. REFERENCES Bryant K. R., Rothwell N. J. and Stock M. J. (1983) Identification of two mitochondrial GDP-binding sites in rat brown adipose tissue. Biosci. Rep. 3, 589-598. Bryant K. R., Rothwell N. J. and Stock M. J. (1984) Acute influences in the two GDP binding sites in brown adipose tissue mitochondria. Biosci. Rep. 4, 5233533. Carey F. G. (I 982) A brain heater in the sword fish. Science 216, 1327-1329. Dawson T. J. and Hulbert A. J. (1970) Standard metabolism, body temperature, and surface areas of Australian marsupials. Am. J. Ph.rsiol. 218, 123331238. Fleming D.. Cinderev R. W. and Hedrn J. P. (1983) The reproductive biology of the Bennett’s wallaby (Macropus r~fogriseus rufogri.seu,s) living fret at Whipsnade Park. J. Zool., Lo&. 201, 283329 I. Foster D. 0. and Frydman M. L. (1978) Nonshivering thermogenesis in the rat. II. Measurements of blood flow with microspheres point to brown adipose tissue as the dominant site of the calorigenesis induced by noradrenaline. Can. J. Physioi. Pharmacol. 56, 110~122. Girardier L. (1983) Brown fat: An energy dissipating tissue. In Mammalian Thermogenesis (Edited by Girardier L. and Stock M. J.), pp. 50-98. Chapman & Hall, London. Mavnes G. (1976)Growth of the parma wallaby, Macropus phrma Waterhouse. Austr. J. .&I. 24, 217-236. Morrison P. and Petajan J. H. (1962) The development of temperature regulation in the opossum, (Didelphis marsupialis oirginiana). Phq’siol. Zool. 35, 52-65. Nicholls D. G. and Locke R. (I 983) Cellular mechanisms of heat dissipation. In Mammalian Thermogenesis (Edited by Girardier L. and Stock M. J.). pp. 849. Chapman & Hall, London. Oliphant R. L. W. (1983) First observations of brown fat m birds. Condor 85, 350-354. Rothwell N. J. and Stock M. J. (1979) A role for brown adipose tissue in diet-induced thermogenesis. Nature 281, 31-35. Rothwell N. J. and Stock M. J. (1984) Brown adipose tissue. In Recent Adwnces in Pll,sio/ogy (Edited by Baker P. F.). Vol. 10. pp. 208233. Churchill Livingstone, Edinburgh.
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