Metabolically functional brown adipose tissue can be pharmacologically stimulated

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1 J. Physiol. (1981), 314, pp With I text figure Printed in Great Britain THERMOGENESIS IN NORMAL RABBITS AND RATS: NO ROLE FOR BROWN ADIPOSE TISSUE? BY J. M. BROCKWAY AND G. E. LOBLEY From the Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB (Received 7 July 1980) SUMMARY 1. The occurrence of dietary and cold-induced thermogenesis in young rabbits was unaffected by noradrenaline or propranolol, and it is concluded that the brown adipose tissue, although detectable histologically, is non-functional. 2. Noradrenaline treatment caused an increase in oxygen consumption in albino, but not in hooded rats, suggesting that the former breed may possess brown adipose tissue capable of thermogenesis. INTRODUCTION The brown adipose tissue of the new-born of many species, and of the adult of some species, is recognized as an important organ for temperature regulation through its capacity to produce a non-shivering thermogenesis (Lindberg, 1970). More recently Rothwell & Stock (1979) have demonstrated that brown adipose tissue is also the site for a dietary-induced thermogenesis in rats consuming an excessive intake of energy when offered a highly palatable diet (Sclafani & Springer, 1976). This so-called cafeteria-fed rat, arguably, does not represent the normal animal, although it may well have some dietary analogies with Western man. It would be of considerable interest to know whether brown adipose tissue is active in the normal animal and thus contributes towards the specific dynamic effect. Hull & Hardman (1970) reported that by one week of age the brown adipose tissue of the rabbit had become functionally altered from its neonatal, non-shivering thermogenic role; but at least until 10 weeks of age the rabbit still has cervical and interscapular fat pads which are a characteristic pale yellow-brown colour. Metabolically functional brown adipose tissue can be pharmacologically stimulated by noradrenaline and inhibited by propranolol. The experiments reported here were an investigation of the response of the brown adipose tissue to these drugs, primarily in young rabbits, and secondarily in two breeds of normally fed rats. METHODS Animal. New Zealand White, Californian and New Zealand White x Californian hybrid rabbits (aged between 3 and 10 weeks and weighing g) and adult male Sprague-Dawley (Department of Zoology, University of Aberdeen) and hooded Rowett strain rats (weighing approximately 350g) were used. The animals were fed on appropriate small-animal stock diets /81/ $07.50 ( 1981 The Physiological Society

2 86 J. M. BROCKWA Y AND G. E. LOBLEY Solutions. (1) Sterile saline solution: 09 % NaCi. (2) 'Levophed' noradrenaline acid tartrate (Winthrop Laboratories), diluted with saline and expressed as weight of base infused. (3) 'Inderal' propranolol hydrochloride (Imperial Chemical Industries), diluted and expressed as weight of salt infused. The drugs were administered i.v. to the rabbits via a peripheral ear vein either as a continuous infusion or as a single injection. Administration of drugs to the rats was by single subcutaneous injections. Before, and between, each drug treatment saline solution was given by the appropriate route, to establish control values. Oxygen consumption. This was measured in an open-circuit respiration apparatus with a 'Servomex' paramagnetic oxygen analyser (range % oxygen). Ventilation rates of the animal chamber were maintained at 81. min' for the rabbits and 41. min- for the rats. Measurements were made over periods of at least 30 min on each drug or control treatment. Except during cold exposure all measurements were made at ambient temperatures of 'C. Body temperature. This was measured with copper/constantan thermocouples calibrated to 0. 1C. RESULTS Histology. The yellow-brown cervical and interscapular fat pads of the young rabbits were of distinctly different colour from the fat tissue from the other parts of the body (e.g. the inguinal region). Under both light and electron microscopes the 'brown' fat pads showed considerable histological differences from normal adipose tissue. The posterior, upper and lateral cervical pads (Hull & Segall, 1965) appeared similar in structure to the classical brown adipose tissue seen in the new-born rabbit (Hull, 1966), while the interscapular pads contained a mixture of brown and white adipocytes. The brown adipose tissue consisted of tightly packed cells in which clusters of large triglyceride droplets occupied approximately 90 % of the cell volume. In the remaining cytoplasm there were numerous large mitochondria with extensive cristae surfaces. Rabbit experiments Experiment 1. The first experiment was to examine whether noradrenaline caused an increase in the oxygen consumption of four conscious rabbits, fed ad libitum. Noradrenaline was infused at dose rates of 2-5, 6-25, 12-5 and 25-0 jg min', but no differences were observed in oxygen uptake between adjacent periods ofnoradrenaline and saline infusion, nor between the different dose levels of noradrenaline. Experiment 2. The possible role of brown adipose tissue as a contributor to the specific dynamic effect (SDE) was studied by administering propranolol during the period of SDE following eating. Four rabbits were used for this experiment, one of which had also been used in Expt 1. They had been accustomed for the previous 7-10 days to a once-daily feeding regime. After removal of food, but during the period when SDE was still manifest, two of the rabbits were given an infusion of propranolol (25,g min-') and the other two an injection (1 mg) of propranolol. A well-marked specific dynamic increase in oxygen uptake was observed for all animals, but neither its time course nor its magnitude were perturbed by treatment with propranolol (Fig. 1). Experiment 3. This was to investigate whether brown adipose tissue contributed to the increase in metabolic rate during cold exposure. Four conscious rabbits, fed ad libitum, were used and their oxygen uptakes measured individually for successive 2 hr periods at temperatures of + 20 and + 5 'C. Cold exposure during saline

3 BROWN FAT THERMOGENESIS 87 treatment increased oxygen consumption by more than 20 %, but injection with propranolol (3 mg) either shortly before (two rabbits) or during (two rabbits) the cold exposure did not reduce the metabolic response to low ambient temperature. Typical values for oxygen consumption of a 600 g rabbit were 18-1 ml. min' at 20 'C and 23'9 ml. min- at 5 'C following injection of saline, and 25-6 ml. min- at 5 0C after administration of propranolol. 0*4 :e 03.2 o mg propranolol i. Food offered.i I I I Time (min) Fig. 1. Changes in oxygen concentration deficit between ingoing and exhaust air from the open-circuit respiration chamber during the SDE, which was not reduced by the administration of propranolol. Mean values from two rabbits. Experiment 4. Four rabbits were anaesthetized with sodium pentabarbitone and thermocouples were placed subcutaneously in the cervical region, adjacent to the brown fat pads, and in the rectum. Anaesthesia was maintained and noradrenaline was infused at 1-25, 2-5, 6-25, 12-5 and 25 Ojug min- but without iany effect on body temperature at either site. Rat experiments In all cases the animals were fed ad libitum, and noradrenaline and propranolol dose levels were 200/jg and 1 mg respectively. Experiment 5. Three Sprague-Dawley (albino) and two hooded Rowett rats were given, on three separate occasions each, injections of noradrenaline and their oxygen uptakes were measured. The Sprague-Dawley rats showed an increased oxygen consumption after injection. This increase was variable, both within and between rats, but averaged 25 % (range 7-38 %) above the levels of oxygen uptake following saline injection. The hooded Rowett rats showed no response in oxygen consumption after treatment with noradrenaline. Experiment 6. A further three Sprague-Dawley rats were treated with (a) noradrenaline and propranolol injected within 5 min of one another, (b) noradrenaline followed 30 min later by propranolol, or (c) propranolol only. When both drugs were

4 88 J. M. BROCKWAY AND G. E. LOBLEY administered within 5 min ofone another no changes in oxygen uptake were observed, regardless of the order in which they were given: mean oxygen consumption for three rats was 9-22 ml. min- after saline and 9 07 ml. min' after the drug injection. In the same rats noradrenaline alone caused a mean increase in oxygen consumption of 34 % ; administration of propranolol, 30 min later, rapidly abolished this increase. Propranolol alone caused no significant diminuation in oxygen uptakes; these were, on average, 9-61 ml. min- and 9-32 ml. min- after saline and propranolol injections respectively. DISCUSSION We conclude from our experiments that, despite its histological appearance the brown adipose tissue of the young, normally fed rabbit does not produce a functional dietary- or cold-induced thermogenic response. This situation is in contrast with that found for the cafeteria-fed albino rat (Rothwell & Stock, 1979). Brown fat thermogenesis in the cafeteria-fed rats may represent an adaptive response to excess dietary energy intake, but both the rabbits and the hooded rats differed from the normally fed albino rats in the present experiments and in those of Rothwell & Stock (1979) in that they did not respond to noradrenaline. This suggests that normal albino rats differ from rabbits and hooded rats in having functional brown adipose tissue, the thermogenic potential ofwhich can be enhanced by an excess energy intake as in cafeteria feeding. Rolls, Rowe & Turner (1980) have commented on the difference between the two breeds of rat in their reaction to regulatory challenges related to persistent obesity, and Rothwell & Stock (1979) have mentioned strain differences in metabolic response within the Sprague-Dawley breed with rats obtained from different sources. Our results confirm, qualitatively, the finding of Rothwell & Stock (1979) that control albino rats increased their oxygen uptake following noradrenaline administration, although the response of the albino rats in the present experiment was only about half of that found by those workers, and was much more variable. Neither the normally fed rabbit nor the albino rat shows a response to propranolol alone: this contrasts with the cafeteria-fed rat, which shows a significant reduction in oxygen consumption after treatment with that drug. However, the normally fed albino rats in this study did respond to propranolol after metabolic stimulation by noradrenaline, strongly reinforcing the suggestion that these rats possess functional brown adipose tissue. It would seem that the presence of brown adipose tissue in an animal does not necessarily mean that the tissue is thermogenically functional. There appears to be a gradation in thermogenic function of brown adipose tissue in the commonly used laboratory small animals, from the rabbit and hooded rat, which show no thermogenesis, through the normally fed, to the cafeteria-fed, albino rat. Dr T. M. King prepared and examined the histological material, and his contribution to this work is gratefully acknowledged.

5 BROWN FAT THERMOGENESIS 89 REFERENCES HULL, D. (1966). The structure and function of brown adipose tissue. Br. med. Bull. 22, HULL, D. & HARDMAN, M. J. (1970). Brown adipose tissue in newborn mammals. In Brown Adipo8e Ti8sue, ed. LINDBERG, O., pp New York: American Elsevier. HULL, D. & SEGALL, M. M. (1965). The contribution of brown adipose tissue to heat production in the new-born rabbit. J. Phy8iol. 181, LINDBERG, 0. (ed.) (1970). Brown Adipo8e Tissue. New York: American Elsevier. ROLLS, B. J., ROWE, E. A. & TURNER, R. C. (1980). Persistent obesity in rats following a period of consumption of a mixed, high energy diet. J. Phy8iol. 298, ROTHEWELL, N. J. & STOCK, M. J. (1979). A role for brown adipose tissue in diet-induced thermogenesis. Nature, Lond. 281, SCLAFANI, A. & SPRINGER, D. (1976). Dietary obesity in adult rats: similarities to hypothalamic and human obesity syndromes. Phy8iol. Behav. 17,