Diet-induced thermogenesis

Phurmac. Ther. Vol, 17, pp, 251 to 268. 1982

0163-7258'82020251-18S09.00,0 Cop),right © 1982 Pergamon Press Lid

Printed in Great Britain. All rights reserved

Specialist Subject Editors: E. SCHONBAtJM a n d P. LOMAX

DIET-INDUCED

THERMOGENESIS

N. J. ROTHWELL*,M. J. ST(XTK* and D. STRIBLING$ *Department q[ Physiology, St. Geor~te's Ho.wital Medical School, Tooting, London SW I 7, U.K. tl.C.l. Pharmaceuticals Division, AIderley Park, Macclesfield, Cheshire, U.K.

1. INTRODUCTION The term thermogenesis strictly applies to all forms of heat production, but the fasting metabolic rate at thermoneutrality (i.e. 'basal' heat production) can be considered as obligatory and therefore distinct from other adaptive (or facultive) forms of thermogenesis. This second category includes thermoregulatory forms of heat production--i.e. shivering and non-shivering thermogenesis (NST), as well as the changes in heat production associated with the utilization of dietary energy diet-induced thermogenesis (DIT). The energy cost of nutrient absorption, transport and assimilation, together with the energy cost of synthetic processes, is one source of DIT but as food intake rises above maintenance requirements there is a disproportionate increase in thermogenesis that serves to dissipate energy consumed in excess of requirements. This adaptive form of DIT shares many of the features of NST but has, until recently, received much less attention than the thermoregulatory component of heat production. NST is particularly important in neonates, hibernators and small rodents (Smith and Horwitz, 1969; Himms-Hagen, 1976) but also has been observed in larger adult mammals (e.g. pig, Kaciubo-Uscilko and Ingram, 1977; calf, Alexander et al., 1975; and man, Jessen, 1980). The importance of DIT and its role in the regulation of energy balance has proved more controversial, although a number of experiments dating back to the turn of the century has demonstrated that in animals (Miller and Payne, 1962; Stirling and Stock, 1968) and man (Neumann, 1902; Gulick, 1922; Miller et al., 1967; Sims et al., 1973), hyperphagia is accompanied by increase in metabolic rate which reduces food conversion efficiency and fat deposition. The failure of many to accept this phenomenon has been largely due to a lack of controlled energy balance experiments and a reliance on indirect estimates of body composition, particularly in man. However, the recent development of a non-stressful model of hyperphagia in the rat has allowed careful study of energy balance and hyperphagia. Rats offered a varied and highly palatable 'cafeteria' diet (comprising of different human food items each day, see Rothwell and Stock, 1979a for details), similar in composition to a human diet, overeat by about 80'~,, compared to control animals fed a stock diet. The hyperphagia produces large increases in energy expenditure and thus the animals avoid excessive weight gain and obesity (Rothwell and Stock, 1979b). When less marked increases in energy intake are achieved, systematic errors complicate the interpretation of data, and it is also difficult to demonstrate DIT in rat strains with a predisposition to obesity. Hyperphagia and DIT are greater in cafeteria rats housed in the cold (Rothwell and Stock, 1980) and it is likely that high environmental temperatures (above thermoneutrality) will inhibit both responses. The demonstration of DIT in the rat has allowed detailed study of the metabolic response to hyperphagia, which has revealed that DIT and NST involve similar physiological and biochemical mechanisms. 2. MECHANISMS OF DIT AND NST Chronic cold exposure causes changes in the levels of several circulating hormones and neurotransmitters but it seems that non-shivering thermogenesis can be directly attri251

252

N.J. R()III\VIII. \t..I. S~oc ~: and I). SIRII31 I\C,

buted to increased sympathetic activity (see Smith and Horwitz. 1969: Himms-Hagen. 1976: Gale. 1973 for reviews). Cold adapted rats have an increased turnover and urinary excretion of noradrcnalinc and show an enhanced thermogenic response to catecholamines. The high metabolic rate of these animals can bc inhibited by' fl-adrcnergic blockade. Although the sympathetic nervous system has been accepted as a mediator of NST for many years, the efl'ector tissue has been the subject of some debate. In neonates and hibernators it was assumed that brown adipose tissue (BAT) was responsible tbr the high rates of heat production, but in adult non-hibernators, where the total mass of BAT usually amounts to less than 2,,, of body weight (Afzelius. 1970), this mechanism was thought to be of relatively' minor importance. This view was reinforced by meast, rements of blood flow using rubidium which showed an apparent decrease in tlow to BAT when NST was stimulated. However. the elegant blood flow studies of Foster and krYdman (1978) in adult cold adapted rats demonstrated that during maximal noradrenaline stimulation the blood flow to BAT. as measured by radioactive microspheres, is equivalent to one third of the cardiac output, and from measurements of oxygen extraction by BAT. they' calculated that this tissue could account for over 60", of NST. "l"hcse studies have therefore established that in the rat. BAT is the most important effector tissue for NST in the body'. Almost all of the changes characteristic of cold adapted animals can be observed in rats exhibiting DIT. Cafeteria fed rats exhibit high metabolic rates which are inhibited by' administration of fl-adrenergic antagonists (Rothwell and Stock. 1979b), increased noradrenaline turnover (Landsberg et al., 1981}. an enhanced thermogenic response to noradrenaline, and hypertrophy and hyperplasia of BAT (Rothwell and Stock. 1979b; Tulp e,' a/., 1980). Blood t]ow studies in these animals have demonstrated that the enhanced thcrmogcnic capacity can be entirch' attributed to the increased oxygen consumption of BAT (Rothwell and Stock, 1981a). These data have led to the proposal that NST and DIT operate via the same mechanisms and this suggestion is supported by the report of improved cold tolerance in cafeteria fed rats and a greater thermogenic response to hyperphagia in cold adapted animals (Rothwell and Stock. 1980: Rothwell et al., 19821. Thermogenesis in BAT could result from either a high rate of mitochondrial oxidation with poor coupling of ATP synthesis, or normal mitochondrial function and increased breakdown of ATP. It is likely that both these mechanisms contribute to the high heat production of BAT. Mitochondria prepared from BAT are usually poorly coupled {Smith et al.. 19661 and it was assumed that this was due to the effects of free-fatty acids released during preparation of the mitochondria. However, Rafael et al. (1969) showed that recoupling of oxidative phosphorylation could be achieved by addition of purine nucleotides, and Nicholls {1979) has proposed that BAT initochondria possess a unique proton conductance pathway which dissipates the proton gradient generated by respiration. This pathway, in the inner mitochondrial membrane, is distinct from the atractyloside-scnsitive uptake pathway and is inhibited by the binding of purine nucleotides (guanosine and adenosine diphosphates, G D P and ADP) to a protein of molecular weight 32.000 Daltons. Nicholls (1979) has also shown that the proton conductance of the inner mitochondrial membrane is proportional to the number of G D P binding sites. Thus, the activity of the pathway can be estimated from the binding of G D P to mitochondria, the effect of purine nucleotides on mitochondrial respiration or the concentration of the 32.0(0} Dalton protein. In fact the high rates of respiration of mitochondria from cold adapted (Nicholls, 1976: Ricquier et al., 1979) and cafeteria fed rats (Brooks e,' oh. 1980) arc markedly inhibited by the addition of purine nucleotides, and specific binding of G D P is increased in NST (Nicholls. 1979: Sundin and Cannon, 1980) and [)IT (Brooks et al., 1980). From Scatchard analysis, it seems that the increased G D P binding rest, Its from an increased number of high affinity binding sites. Furthermore. a reduction in G D P binding in BAT has been observed in genetically obese mice (Himms-Hagcn and Dcsautels, 1978: G o o d b o d y and Trayhurn, 1981) which also have a defective thermogenic response

Diet-induced thermogenesis

253

to diet and cold (Thurlby and Trayhurn, 1979). In spite of these obvious correlations between the activity of the proton conductance pathway and thermogenesis, it is at present impossible to quantify the physiological contribution of this system to metabolic rate in vivo. In addition to changes in mitochondrial coupling, non-productive hydrolysis of ATP could also be responsible for heat production in BAT. Horwitz (1979) has suggested that increased activity of the membrane bound sodium potassium adenosine triphosphatase (Na+,K+-ATPase) may be involved in BAT thermogenesis. The activity of this enzyme and its response to noradrenaline are increased in both cold adapted (Horwitz, 1973) and cafeteria fed animals (Rothwell et al., 1981b) and in the latter case a remarkable correlation has been observed between the Na+,K+-ATPase activity in Htro in BAT and metabolic rate in ritro (Rothwell et al., 1981b). Alexson et al., (1981) have reported increased peroxisomal fl-oxidation in BAT from cold adapted rats. However, although this process would lead to an increase in uncoupled fl-oxidation of fatty acids, it is quantitatively unimportant in terms of the total heat production of the tissue. Newsholme and Crabtree (1976) have proposed that substrate cycles may be important in heat production. BAT has high maximal rates of lipolysis, lipogenesis and fl-oxidation and therefore futile cycling could result in significant energy wasting via ATP hydrolysis. However, it has been established that triglyceride cycling cannot account for more than l'k,~of the oxygen consumption of brown fat in tritro from neonatal rabbits (Knight and Myant, 1970). BAT also contains significant levels of FDPase which decline with age and sympathectomy, but are increased by cold exposure (Seccombe et al., 1977). However, the activity of the PFK/FDPase cycle declines when BAT is activated by either cold exposure or cafeteria feeding (Stribling, unpublished data).

3. CENTRAL CONTROL OF THERMOGENESIS The hypothalamus acts as an integrator of most basic physiological functions and plays an important role in thermoregulation. In addition to temperature sensitive neurones in the preoptic anterior hypothalamus, which increase firing rates to produce either a rise or fall in local temperature, the hypothalamus receives afferent inputs from central, peripheral and spinal temperature sensors. This information is used to regulate body temperature by modulation of heat loss and thermogenesis (both shivering and NST). In the rat, cooling the preoptic area increases resting oxygen consumption and the effects can be blocked by propranolol (Banet et al., 1978), which has no effect on shivering thermogenesis (Horwitz and Hanes, 1976) indicating a neural connection to BAT. Stimulation of thermogenesis through excessive nutrient intake (DIT) has an inevitable impact on thermoregulation which would involve the hypothalamus, even if only indirectly. However, the ventromedial hypothalamus is known to be involved in appetite and weight regulation. Whilst most lesions of the VM H cause hyperphagia, which in turn has been held as the mechanism of the ensuing obesity, it is possible to generate lesions which do not cause hyperphagia but still cause increased fatness (Rabin, 1974). This implied role in the control of thermogenesis is supported by the observation that stimulation of the VMH increases BAT temperature (Perkins et al., 1981). Furthermore, by three days after lesioning, the BAT of VMH lesioned rats becomes insensitive to nerve stimulation or noradrenaline administration, implying a physiological role of the VMH in acute and trophic control of BAT (Seydoux et al., 1981 ). Lesions or stimulation of the VMH could be affecting a pathway involved with thermoregulation rather than D1T per se. However, in a recent study it was shown that VMH lesioned young rats maintained core temperature on exposure to 5°C and their maintenance energy requirements were unchanged, but the efficiency of energy retention above maintenance was markedly increased (Vander Tuig et al., 1980). This suggests that the VMH is directly concerned with DIT rather than NST.

254

N . J . R{llitWt.Ii, M. J. SIO(K ;lilt] D SIRIH;I I\(,

It is unclear whether the VMH itself contains metabolic receptors or receivcs afferent inputs from other sites. Central injections of 2-deoxy-glucose, which causes cellular gh,copaenia (Brown, 1962}, reduce core temperature in rats (Fricnkel ct ~d., 1972~. Ahhough the ventral premammilary nucleus appears to be particularly responsive, hypolhermic effects are also obtained from injections into other hypothalanlic sites (Shiraishi and Mager. 1980). The region of the VMH which affects DIT does appear to have a particularly high rate of insulin-dependent glucose utilization since parentcral injection of gold thioglucose in mice deposits gold in the hypothalamus and achieves parallel functional changes to an electrolytic lesion in the VMH. (Debons et al.. 1968, 1970}. Panksepp (1974) has shown that direct administration of glucose into the VMH induces satiety and this is dependent upon incorporation of the glucose into the tissue, but there are as yet no reports of effects of central administration of nutrients on thermogenesis or activity of BAT. However, insulin increases turnover of monoamines in the ra! hypothalamus (Sauter et al.. 1981) and gold thioglucose treatment of mice disrupts the dietary regulation of sympathetic activity without affecting sympathetic activation by cold exposure (Young and Landsberg, 1979} which suggests the presence of nutrient receptors m this region. It would be easy to postulate a simplistic theory of hypothalamic control of BAT. However, the innervation of BAT is complex (Flaim et al.. 19761 and there is amplc evidence for the existence of other control systems. Just as thermoregulation is dependent on ascending and descending facilitatory and inhibitory pathways between the CNS. hypothalamus and brain stem, it is likely that central regulation of DIT will prove to be a complex integration of afferent and efferent signals coupled with control of appetite, behavior and peripheral metabolism.

4. NEUROENDOCRINE CONTROL OF THERMOGENESIS 4.1. CATECHOLAMINES Cold exposure results in a dramatic increase in the urinary excretion of catccholamines and their metabolites in many species, including man (see Himms-Hagen. 1967; Gale 1973, for reviews). The rise in catecholamines (predominantly noradrenaline and adrcnaline) is due to both an increase in adreno-medullary secretion and a high ratc of release of noradrenaline from sympathetic nerves (see Himms-Hagen, 1975 for revicw). Both of these systems are involved in the response to cold. but it seems that neural rclcase of noradrenaline is primarily responsible for NST. It is difficult to establish the relative importance of the adrenal medulla and sympathetic nerves since ablation of one is oflcn accompanied by a compensation in the other. However. adrenal demcdullation causes only slight impairment of thermogenesis while immunosympathectomy causes hypothermia and impaired cold tolerance in young rats but less marked effects m older animals (see Himms-Hagen, 1975 for review). Furthermore. the concentrations of exogenous adrenaline and noradrenaline required for maximal stimulation of metabolic rate (Rothwell and Stock, 1979b), BAT temperature (Flaim et al., 1977) or GDP binding (Nicholls, 1979; Brooks et al., 1980) are far in excess of circulating levcls, indicating that BAT thermogenesis results largely from activation of the sympathetic nerves and synaptic release of noradrenaline. Studies of adrenergic receptors which mediate depolarisation of brown fat in neonatal rats (Fink and Williams, 1976), indicate that noradrcnalinc, phenylephrine and isoproterenol all depolarise brown adipocytes to a similar extent. Propranolol competitively inhibits depolarization induced by isoproterenol, confirming involvement of the fl-receptor, whereas the effects of phenylephrine arc inhibited by the z-blockers phcntolamine and phenoxybenzamine. Activation of the membrane Na+,K'-ATPase by noradrenalme is, almost totally blocked by propranolol (Rothwell et al.. 1981b) indicating that this physiological response is mediated via the fl-receptor.

255

Diet-induced thermogenesis TABLE 1. Role of the fl-receptor in Noradrenaline Stimulated Increase in GDP Binding of B A T Mitochm,dria Antagonist (mg/kg, sc) Saline Propranolol (20) Phentolamine (20) Saline

Agonist (mg/kg. sc) Noradrenaline (0.5) Noradrenaline (0.5) Noradrenaline (0.5~ Saline

G D P binding* (nmol/mg) (I.314 0.178 0.310 0.192

P

+ 0.047 _+ 0.008 _+ 0.011 _+ 0.017

< 0.02 NS <0.05

Mitochondria were prepared from rats housed at 24 C, dosed with antagonist 30 min before noradrenaline and BAT removed 60 min later. G D P binding was determined by method of Nicholls (1976). *Mean values + SEM. n = 6. IDavidson and Stribling, unpublished data.)

Brown fat cells of the hamster have a high density of specific, high affinity (3HI-dihydroalprenolol binding sites (Svoboda et al., 1979) and the relative potency of adrenergic agonists in displacing the (3H)-dihydroalprenolol indicated a fit-receptor sub-type. The same rank order of potency was obtained for stimulation of oxygen consumption, indicating the functional activity of the fix-receptor. Administration of noradrenaline to rats causes a rapid increase in the specific G D P binding of mitochondria subsequently isolated from BAT. The increase in G D P binding can be totally prevented by pretreatment with propranolol (Table 1). However, neither the selective fix-antagonist atenolol, nor a selective fl2-antagonist ICI 118551, completely prevents the response, although in combination they achieve a total blockade (Table 2). This indicates a mixed flx/fl2 receptor population mediating the mitochondrial response to noradrenaline in vivo, and is supported by the observation that clenbuterol (a selective fiE stimulant) increases G D P binding (Table 3). There is some evidence, however, to suggest that ~-adrenoreceptors are also involved in thermogenesis. Membranes prepared from hamster BAT bind the non-selective ~-antagonist (3H)-dihydroergocryptine and a high proportion of this can be displaced by the ~-specific ligand, phentolamine (Svartengren et al., 1980). The fl to ~-receptor density, TABLE 2. flr"fl2 Selectivity of the Noradrenaline Stimulated Increase in GDP Binding Of B A T M it ochondria. Antagonist (mg/kg, sc) Saline Atenolol [20) ICI I18551 (20) Atenolol (20) + ICI 118551 (20) Saline

Agonist (mg/kg, sc)

G D P binding* (nmol/mg)

P

Noradrenaline[0.5) Noradrenaline (0.5) Noradrenaline(0.5) Noradrenaline (0.5)

0.290 0.208 0.240 0.147

_+ 0.012 +_ 0.006 '_- 0.009 + 0.009

< 0.001 <0.001 <0.001

Saline

0.150 + 0.011

<0.001

*Mean values + SEM, n = 6 (Davidson and Stribling, unpublished data.) TABLE 3. Role of the fl2-Receptor in the Control of GDP Binding o) B A T Mitochondria Antagonist (mg/kg, sc) Saline Atenolol (20) 118551 (20) Saline

Agonist (mg/kg, sc) Clenbuterol(l} Clenbuterol ( 1) Clenbuterol(1) Saline

G D P binding* (nmol/mg) 0.323 0.257 0.182 0.141

4- 0.019 _+ 0.014 _ 0.010 +_ 0.007

P < 0.002 <0.001 <0.001

For details see legend to Table 1. *Mean values + SEM, n = 6 (Davidson and Stribling, unpublished data.) JP.T

17 2- H

25(>

N.J. R())))wH.I., M. J. Sr()('K and D. S~'Rmt I',;(; TA))I.t 4. Role ol tire z~-R(,ceptor in the Control u! (iDP Bimlimt ol BAI" .~,litoclumdria. Antagonist Img kg, so)

Agonist (mg kg. so)

Saline Atcnolol(20) Phentolaminc (20) Saline

Phenylephrinc (21 Phenylcphrine(2) Phcnylephrinc (2) Saline

(}DP bindin;g (nmol mg) 0.37~ 0.298 0.225 0.182

-,_-

0.016 0.015 0.009 0.007

/'

<0.01 < 0.00t <0.001

For details see legend to Table I. (1)avidson and Stribling. unpublished data.)

however, is in the ratio of 5:1 {Svartcngren et al., 1981). The oz,-receptor is functional, since stimulation of isolated BAT cells from rats with adrenaline causes an activation of phosphatidyl inositol labelling which is inhibited by the selective ~l-antagonist prazosin (Garcia Sainz et al., 1980). Administration of phentolamine to rats does not affect the increase in GDP binding induced by exogenous noradrenalinc {Table 1) but the ~-agonist phenylephrine causes a partially/3-receptor independent increase in mitochondrial GDP binding {Table 4). This is consistent with the observation that phenylephrine causes a significant increase in metabolic rate in both control and cafeteria fed rats (Fig. 1), but this response is much larger in the former group. The effects of phenylephrine on Na+,K "-ATPase activity in vitro are, however, greater in tissue from cafeteria fed rats and some 3()°4~of the response to noradrenaline is dependent on ~-receptor systems. Although almost 90'~'o of the elevated metabolic rate of cafeteria fed rats is abolished by /~-adrenergic blockade with propranolol, (Rothwell and Stock. 1979b) phentolamine does have a small (20~o) effect (Rothwell et al.. 1981c). The role of :~-receptors extends beyond direct effects on the BAT cell. For example, Flaim et al. [1976) have studied the effects of electrically stimulating the sympathetic nerves supplying the interscapular BAT depot on the temperature of the tissue. They noted an initial fall in temperature, which was attributed to vasoconstriction and was abolished by phentolamine. This was followed by a large rise in temperature apparently due to increased heat production by BAT. which was abolished by propranolol but unaffected by phentolamine. In addition to various post-synaptic adrenergic receptor systems, the activity of BAT can be modulated by increasing the synaptic concentration of noradrenaline by blocking its reuptake with, for example, ciclazindol. This drug increases metabolic rate in rats and

2O /

/

/ /

/

I

E

T

J/

s / •

j /

//

// // Controt

Cofeterio

FIG. 1. Resting oxygen consumption of control and cafeteria fed rats before (open bars) and after (hatched bars) injection of phenylephrine (5 mg,:kg b.wt., s.c.). Mean values, n = 6 bar denotes SEM. (Rothv,ell, Stock and Winter. unpublished data.)

257

Diet-induced thermogenesis TABLE 5. Role of the ~t2-Receptor in the Control of

GDP Bindin9 of BAT Mitochondria. Agonist (mg/kg. sc) Saline Yohimbine (1) Noradrenaline(0.5)

G D P binding* (nmol/mg)

P

0.23 _+ 0.01 0.30 _+ 0.01 0.30 _+ 0.01

<0.01 <0.01

For details see legend to Table 1. *Mean values + SEM n = 6 (Davidson Stribling. unpublished data.)

and

the effect involves increased activity of BAT (Rothwell et al., 1981c). A similar sympathomimetic effect on BAT GDP binding can be achieved with the ct2-receptor antagonist, yohimbine (Table 5). There are two possible types of 22-receptor involvement; a postsynaptic site inhibiting cAMP accumulation in response to a//-stimulus as seen in white fat cells (Garcia Sainz et al., 1980) or a presynaptic site operating a negative feed back mechanism on further neurotransmitter release (Langer, 1977). In both cases, the end effect would be dependent on a primary fl-stimulus, but in the latter case this would be accompanied by an increased turnover of noradrenaline. Thus, although the primary effects of noradrenaline in BAT are mediated via a fit-receptor, the results discussed above indicate the possible involvement of f12, :tt and ct2 receptor systems in either the direct or indirect control of BAT thermogenesis. Some of the apparent anomalies could be explained by species differences between the hibernating hamster and the adult rat. Alternatively, the involvement of a certain receptor type in the activation of one component of the thermogenic response does not guarantee an effect on metabolic rate in vivo, nor a role in the physiological response to hyperphagia. Noradrenaline is assumed to exert a dual influence on BAT, by rapidly stimulating substrate mobilization and heat production and, secondly, by inducing a chronic increase in the mass of BAT which is usually due to both hypertrophy and hyperplasia of the tissue. LeBlanc and Villemaire (1970) have demonstrated that twice daily injections of noradrenaline in rats for several weeks produces a marked increase in the mass of BAT and an enhanced acute response to the hormone. Desautels and Himms-Hagen (1979) have also reported BAT hypertrophy after chronic treatment with noradrenaline, but found that the changes in mitochondrial protein composition which result in an increase in proton conductance pathways during cold exposure were not mimicked by noradrenaline treatment. Furthermore, Mory et al., (1978) have noted hypertrophy of BAT in sympathectomized rats exposed to cold, indicating the involvement of a trophic factor other than noradrenaline. However, maintenance of fit-blockade with atenolol does reduce the peak 3H-thymidine incorporation into BAT in rats exposed to 4°C (Table 6). TABLE 6. 3H-thymidine Incorporation into Interscapular BAT

Pre-treatment Saline Atenolol ICI 118551 Not cold cxposed + Saline

Thymidine incorporation* (dpm × 10-3.'IBAT pad) 44.4 23.9 33.6 17.7

+ 4.8 + 3.4 +_ 5.8 + 0.2

% difference

-46.1 -24.3 -96.0

Rats were cold exposed to 4 C for 48 hours and injected (sc) with drugs (20 mg/kg) twice daily at 9.30 and 16.30 and at 9.30 on the final day before administration of 6-3H thymidine (0.5 ,uCi/g body weight). aH-thymidine b o u n d to D N A was determined in BAT removed 4 hr later. *Mean values + SEM, n = 6 (Davidson and Stribling, unpublished data.)

258

N . J . R o l m ' . ' H I.. M. J. St~u~. a n d D. S r a m l ,x(; TABI.I 7. ReMinq I~'O2 and (il)f' Bindin~l Belore and .4lter

I)opamine ,4hme [5mff.k~l~ or Dopaminc Plus (20 m~t.kqt

Resting ~"O2 ( m l / m i n . W ¢~- 5)-iMitochondrial GDP binding (nmol.:mg protein)'l"

h!jection o/ ~-auta.qonist Ipropranolol)

Dopamine - fl-antagonist

Control

Dopamme

12.70 _+ 0.36

17.78 ~- 0.63*

14.62 ± 0.(}5

130 4- g

179 + 4*

159 ± 7*

t M e a n Values -,- SEM, 11 - 6 *P < 0.(X)l C o m p a r e d to Controls. (I')avidson, Rothwell, Stock and Stribling, u n p u b l i s h e d data.)

The third major catecholamine, dopamine, has been implicated in the central control of food intake and body temperature, but recent data indicate direct peripheral effects of this transmitter on thermogenesis. For example, Derry et al. (1969) produced some evidence for dopaminergic interneurones in the sympathetic innervation of BAT cells, whereas blood vessels in BAT are directly innervated. Peripheral injections of the dopamine antagonist pimozide completely abolish the elevated metabolic rate of cafeteria fed rats (Rothwell et al., 1981c) and significantly reduce the high in vitro activity of Na+,K +-ATPase in BAT from these animals (Rothwell et al., 1981c). We have also found that peripheral injections of dopamine produce significant rises in resting 902 and BAT mitochondrial GDP binding, but only the oxygen consumption response is inhibited by fl-adrenergic blockade (Table 7). The turnover rate of dopamine in BAT in vivo is elevated by about 100%; in cafeteria fed rats compared to controls but release in vitro is very low in comparison to noradrenaline (Rothwell, Stock and Wyllie, unpublished data). This, together with the low potency of dopamine relative to noradrenaline indicates only a minor peripheral role for this transmitter in thermogenesis. 4.2. T H Y R O I D H O R M O N E S

Cold exposure and overfeeding produce a rise in plasma thyroid hormones (Reichlin et al., 1973; Rothwell and Stock, 1979b; Tulp et al., 1980) particularly triiodothyronine (T3), and short-term changes in T4/T3 ratio occur postprandially (Hesse et al., 1981). These data have fostered the idea that the thyroid is an important mediator of thermogenesis. Indeed hyperthyroidism and hypothyroidism result in profound increases and decreases in metabolic rate respectively, but the mechanism of these thyroid effects on heat production may differ from those involved in catecholamine induced thermogenesis. Chronic treatment with high doses of thyroid hormones causes an enhanced response to noradrenaline and hypertrophy of BAT (LeBlanc and Villemaire, 1970) but the latter is partly due to a high lipid deposition in the tissue (Lachance and Pag6, 1953), and Sundin (1981) has observed a reduced level of BAT mitochondrial GDP binding in hyperthyroid rats. Surgical or chemical thyroidectomy does result in impaired cold tolerance, which is probably due to a reduced fl-reccptor number, but cold tolerance can be restored by theophylline treatment (Fregly et al.. 1979). Hypothyroid animals still show hypertrophy of brown fat during cold exposure (Ikemoto et al., 1967) and cafeteria feeding (Table 8), and thyroidectomized animals often have a greater mass of tissue and a lower lipid content than euthyroid controls (Saville and Stock, unpublished data). Doniach (1975) has suggested that thyrotropin (TSH) may stimulate the growth of brown fat. She has noted a swelling of the supraclavicular fat pads and hypertrophy of BAT in patients with myxoedema, where TSH levels are very high, and this is reversed by treatment with thyroxine (T4) which reduces TSH levels. However. lack of T4 is not the cause of these features since they are not found in pituitary myxoedema where thyroid hormone levels are low but TSH is absent. Consistent with this suggestion, there is a marked increase in

Diet-induced thermogenesis

259

TABLE 8. lnterscapular BAT Mass (mg/lO0 g b.wt) in Euthyroid, Thyroidectomized (TX) or Thyroidectomized Rats Replaced with T3 (365ng/lOOq b.wt./d) Fed either Stock or Cafeteria Diet Interscapular BAT mass* Control Cafeteria TX control TX Cafeteria TX T3 control TX T 3 cafeteria

84 130 196 239 134 177

_+ 10 + 9 + 12 ___24 + 10 + 14

*Mean values _+ SEM, n = 8 (Saville and Stock. unpublished data.)

TSH release on exposure to cold, which could perform a dual function in stimulating thyroidal secretion of T4 and T 3, and inducing hypertrophy of BAT. Consideration of these findings suggests that the primary role of thyroid hormones (T4 and T3) in thermogenesis is to produce a sensitization of BAT to noradrenaline by increasing the number of/~-receptors and regulating the activity of the adenyl cyclase system. It can be seen from Fig. 2 that the thermogenic response to noradrenaline is reduced in thyroidectomized and elevated in hyperthyroid rats and the sensitivity of BAT Na+,K ÷-ATPase follows a similar pattern. In addition, it seems that catecholamines may stimulate thyroidal release and peripheral conversion of T4 t o T 3. Melander et al. (1972, 1973) have reported a rich sympathetic innervation of the thyroid, stimulation of which causes release of thyroid hormones. It has also been observed that propranolol causes a reduction in s e r u m T 3 levels and a simultaneous rise in concentrations of the inactive isomer r e v e r s e T 3 (rT3), indicating a modification of the pathway for peripheral deiodination (Verhoeven et al., 1977). Chronic injections of noradrenaline in rats result in a significant elevation in circulating T 3 levels and an increase in liver mitochondrial ~-glycerophosphate dehydrogenase (Table 9), an enzyme which is particularly sensitive to thyroid status. Thus, the sensitization to catecholamines by T 3 and a stimulation of T 3 levels by noradrenaline represents a potential positive feedback mechanism whereby a small change in either hormone could result in a greatly amplified effect on thermogenesis. 2B

50O

a. I 8rY 4

ol Control

R TX

Hyper thyroid

40C 300 200

+o z

IO0

nN

Control TX

Hyper thyroid

FIc;. 2. Resting oxygen consumption (ml/min/W °75) before (open bars) and after (hatched bars) injection of noradrenaline (0.25 mg/kg b.wt. s.c.) and in vitro Na ',K "-ATPase activity in BAT (nmol Pi/mg P/h) without (open bars) or with (hatched bars) noradrenaline added to the medium ( I 0 4 M), in control, tbyroidectomized (TX) and hyperthyroid (T3 treated) rats. Mean values, bar denotes SEM (n = 6--10) (Rothwell, Saville, Stock and Wyllie, unpublished data.)

2(,a)

N

J, r ( u i l \ V i 1.1.. M. J, Sro(K and D. SIRIBI Ix;(i

TABI.t 9. lnterscapular 13,1T .'~h:s~. Plasma "I3 Lel el.s and Lil er Mitoctu,ndrial "J-(;i~ ,'ropho',phatc o~idu.se l,et cl~ in Control and :Voradrenalim, 7)'eated Rats i401#/ .Voradremdim' I(X}~/ h.wt. l~ice Daily lot 7 D,.I'~}.

Liver mitochondrial lntcrscapular BAT mass{. Img lOOg b.wt) Control Noradrcnaline

52 z_+ 2 108 _+ 9+

Plasma "I"3{ (rig lOOml) 29 + 3 62 + 11"*

>OPO

activity +

(l~mol mg protein mini 1.02 + 0.20 I.~6 + I).26"

weated *P < 0.05. **I' < 0.01. +P < O.(X)I c o m p a r e d to c o n t r o l s . (Rothwell. Saville a n d Stock. u n p u b l i s h e d d a t a ) ++Mean Values ~- S E M . n = 6.

4.3. INSULIN Insulin is generally considered to be a powerful anabolic hormone because of its stimulatory effects on fat. protein and glycogen synthesis, and the increases in body fat content which result from chronic insulin treatment (Hoebel and Teitclbaum, 1966: Macdonald et al., 1976) suggest that this hormone may inhibit thermogenesis. However. there are also data to indicate that insulin may be required for NST and DIT, and that this hormone may directly stimulate BAT and potentiate the effects of noradrenaline. Diabetic animals fail to exhibit DIT when fed a cafeteria diet unless they are injected with insulin (Rothwell and Stock, 1981b). NST appears to be similarly insulin dependent since Mondon (1963) has demonstrated that alloxan--diabetic rats are prone to hypothermia, whilst insulin treatment reverses the impaired oxygen consumption responsc to cold (Drury, 1957}. It is also interesting to observe that many of the genetically obese rodents (e.g. ob,..'ob and db,'db mice}, which have abnormal insulin responses, also have defective thermogenesis (Cox and Powley, 1977; Thurlby and Trayhurn, 1979; Bray and York, 1979). In young rats, insulin can potentiate the in vivo oxygen consumption response to noradrenaline (Rothwell and Stock, unpublished data) but this was not observed in older animals (Rothwell and Stock 1981b). Therminarias et al. (1979) have also reported a calorigenic effect of insulin and improved resistance to cold in dogs. Similarly, Bennett et al., (1980) have found a 23°I; increase in energy expenditure, which was inhibited by fl-adrenergic blockade, in human subjects during insulin-induced hypoglycemia. The latter observation may be due to the low blood glucose rather than to insulin per se since in man, glucopenia induced by 2-deoxyglucose causes a rise in metabolic rate (Thompson et al., 1980), whereas the reverse occurs in the rat (Shiraishi and Mager, 1980). Insulin can also stimulate BAT directly or potentiate the thermogenic (Dawkins and Hull, 1963; Shackney and Joel 1966) and lipolytic effects of catecholamines (Chinet, A., personal communication) which suggests it has a role in the supply of st, bstrates for catabolism. 4.4. GI,U('AGON Kuroshima's group (Kuroshima et al., 1977: 1978: 1979: Kuroshima and Yahata. 1979: Yahata et al., 1981) has carried out a number of studies on the metabolic effects of glucagon, particularly in relation to BAT, and they conclude that this hormonc is involved in NST. Glucagon stimulates heat production of brown adipose tissue both in t'ivo (Heim and Hull, 1966; Cockburn et al., 1968) and in vitro (Joel, 1966: Friedli et al., 1978) and Kuroshima and Yahata (1979) found that glucagon caused a twofold increase in the heat production of isolated brown adipocytes. The maximal response of brown fat cells to glucagon was greater than the response to noradrenaline, and was reduced in cells prepared from heat acclimatized animals, but was unaltered by cold exposure. Chronic administration of glucagon to rats improves cold tolerance, increases the size of

Diet-induced thermogenesis

261

BAT depots and causes functional adaptation of BAT mitochondria (Yahata et al., 1981). Glucagon also stimulates the metabolic rate of white adipocytes, but in this tissue the response is enhanced by cold exposure and attenuated by heat acclimation. The mechanism of action of glucagon on BAT thermogenesis is not fully understood but may involve stimulation of adenyl cyclase. Hyperthyroidism potentiates glucagon stimulation of cyclic AMP production and lipolysis in association with an increase in glucagon binding sites to fat cells (Madsen and Sonne, 1976), and Kuroshima et al., (1979) suggest that cold and heat acclimation modify adrenergic and glucagon receptors in brown fat cells. However, the effects of glucagon on oxygen consumption of isolated BAT cells, is partially inhibited by /3-blockade (Holloway and Stribling, unpublished data) and it is possible that these effects of glucagon may be due to non-specific interaction with fl-adrenoreceptors. Ingram and Kaciubo-Uscilko (1980) have also found that glucagon stimulation of oxygen consumption in the pig is abolished by fl-adrenergic blockade, although there is no clear evidence that the pig depends on BAT for NST. There is no doubt that exogenous glucagon exerts a marked effect on metabolic rate and brown adipose tissue thermogenesis but the physiological role of glucagon is less certain. A rapid rise in plasma glucagon has been observed in rats and men exposed to cold (Seitz et al., 1981) and higher circulating levels of glucagon have been observed in Japanese farmers during the winter than the summer months (Kuroshima et al., 1981), but the exogenous doses of glucagon required to stimulate thermogenesis are rather high compared to plasma levels. Kuroshima and Yahata (1979) found a maximal stimulation of brown fat cell heat production by glucagon and noradrenaline at a dose of 1 #g/ml, which is three orders of magnitude higher than circulating levels of glucagon but may be comparable to the concentrations of noradrenaline at the synapse after neural release. Nevertheless these authors did observe a doubling of brown adipocyte heat production by lower concentrations of glucagon (0.1 l~g/ml) and the use of proteolytic enzymes in the preparation of the adipocytes can impair receptors for peptide hormones without affecting the/t-receptor. Glucagon has also been implicated in the regulation of body weight. For example, De Castro et al. (1978) have proposed that the ratio of insulin to glucagon levels is a regulator of the set point for body weight and found that chronic glucagon treatment produced a marked depression of body weight. The authors attributed this observation to a 30~o reduction in food intake, but rats can partly compensate for a calorie deficit by an increase in metabolic efficiency and the large depression in body weight may also reflect an effect of glucagon on metabolic rate. 4.5. STEROIDS Deavers and Musacchia (1979) have recently reviewed the role of glucocorticoids in thermogenesis, particularly in relation to lipid and carbohydrate metabolism and conclude that most of these hormones are permissive in their effects. Adrenalectomized animals are unable to maintain a normal body temperature in hot or cold environments, partly because of insensitivity to catecholamines but also because of an inability to activate shivering or improve insulation by piloerection or vasoconstriction (Maickel et al., 1967a). The impairment of thermoregulation is largely due to the absence of the adrenal cortex since replacement with glucosteroids allows restoration of homeothermy. Glucocorticoid treatment also causes an enhanced thermogenic capacity in hypothermic animals which, unlike hibernators, cannot normally arouse from a low body temperature. These hormones are largely permissive in their actions and are required for the lipolytic actions of catecholamines, ACTH and growth hormone, although they also have direct lipolytic effects in white adipose tissue. Removal of the adrenals or of the pituitary leads to a progressive loss of glycogen (Tuerkischer and Wertheimer, 1946) and lipid (Mazzuchelli et al., 1961) in brown adipose tissue which is reversed by cortisone treatment. In intact animals, short term administration of cortisone or ACTH stimulates lipid and glycogen deposition and causes an

262

N J. RoNJwl!t.t.. M. J. STo('rz a n d D. S r , mt ix~i

increase in the mass of BAT (Lachance and Pag6, 1953), though the response to ACTH is absent in adrenalectomized animals indicating that the effect is mediated by release of glucocorticoids. The lipogenic effect of these hormones contrasts with the lipolytic responses observed in white fat and may be secondary to elevated glucose levels made available to brown fat from other sources (Mazzuchelli et al., 1961). However. BAT cells do possess specific dexamethasone receptors and dexamethasone inhibits uridine incorporation into TCA precipitable material (Feldman, 1978) indicating that BAT is a glucocorticoid target organ. Cold exposure usually results in hypertrophy of the adrenal cortex and an increase in plasma cortisol levels in animals (Collins and Weiner, 1968; Panaretto and Vickerey, 1970) and man (Wilson et al., 1970) although it is uncertain whether this has any calorigenie influence on thermogenesis. Smith and Horwitz (1969) concluded that ACTH and glucocorticoids may influence BAT thermogenesis by affecting the amount of substrate (lipid), the oxygen supply to the tissue and the convective heat loss. Jansky (1973) has noted a 25'~o increase in metabolic rate during ACTH infusion which was not affected by fl-blockade. Laury and Portet (1977) were unable to demonstrate an acute effect of ACTH on metabolic rate or the response to noradrenaline in normal rats, but after chronic administration of ACTH (2lU/kg:'day for 10 days) basal metabolic rate increased by 20% and the calorigenic effect of noradrenaline was increased by 50"0. This is consistent with the observation that administration of ACTH to rats (11U..,'kg/day for 14 days) causes a significant increase in BAT mitochondrial G D P binding. Since the glucocorticoid triamicinolone (25/~g.."kg/day) was without effect in the same system (Stribling and Davidson. t, npublishcd data), this may indicate that the glucocorticoids and ACTH exert independent direct effects on thermogenesis. The pattern of effects seen in normal rats is not consistent with those reported for the ob.Ob mouse. Although hypothalamic levels of corticotropin releasing factor are normal in this mutant, pituitary levels and release of ACTH are elevated (Edwardson and Hough, 1975). The adrenal glands are also larger (Marshall et al., 1957), and circulating levels of corticostcrone higher (Naescr, 1974). Adrenalectomy prevents excess weight gain in this animal, independent of effects on food intake (Yukimura and Bray, 1978) which implies the removal of an inhibiting influence on thermogenesis. There are a number of reports indicating effects of the sex steroids on body weight. which cannot be directly related to changes in food intake. Ovariectomy results in excessive weight gain in rats which is inhibited by replacement treatment with estrogen or estradiol (Laudenslager et al., 1980}. Normal animals given oestrogen or testosterone show reduced or increased rates of weight gain respectively (Earley and Leonard. 1979). These variations in body weight arc accompanied by changes in food intake but thcrc is also evidence for marked changes in the efficiency of food utilization. The weight gains induced by testosterone (Earley and Leonard, 1979) or ovariectomy (Kanarck and Beck, 1980) are associated with an increased efficiency of weight gain, while estrogen treatment causes a reduced efficiency (Earley and Leonard, 1979) and a significant increase in heat production (Laudenslager et al., 1980). We have found that a single injection of oestradiol produces only a very small increase in metabolic rate (8-- 1004,) and no change in BAT temperature in the rat (Rothwell and Stock unpublished data) but the above results all relate to chronic treatments indicating that any thermogenic response to these steroids may require some time to develop. Cyclic variations in body temperature associated with the ovarian cycle (Marrone et al., 1976; Moghissi, 1976) have also been related to varying levels of estrogen and progesterone but it seems that these may be due to a central action of the steroids on the set point for body temperature regulation rather than to changes in peripheral heat production (Gale, 1973). 4.6. OmER FACTORS It is impossible in this review to consider all of the other hormones which have at

Diet-induced thermogenesis

263

some time or another been implicated in the central or peripheral control of thermogenesis and we will therefore discuss only those for which there is some experimental evidence of thermogenic effects. Prostaglandins injected in very small amounts into the hypothalamus produce a rapid rise in body temperature (Stitt and Hardy, 1972) and prostaglandin synthetase inhibitors will reduce body temperature during fever. It has been claimed that prostaglandins are not involved in the normal regulation of body temperature or in the thermogenic response to cold since prostaglandin synthesis inhibitors do not affect temperature in these situations (Cranston et al., 1970). Similarly, chronic treatment with prostaglandin synthetase inhibitors does not cause any significant change in body weight (Kather et al., 1978). Nevertheless we have observed that peripheral injection of the prostaglandin inhibitor flurbiprofen completely abolishes the increased metabolic rate of cafeteria fed rats but has no effect"on the oxygen consumption of stock fed control animals (Rothwell et al., 1981¢'). Sympathetic stimulation of white fat is associated with release of prostaglandin E1 which inhibits lipolytic responses, (Shaw and Ramwell, 1968; Effendic; 1970). However, no effects of prostaglandins on BAT have been reported. Inhibition of prostaglandin formation in white fat during lipolysis prevents vasodilatation and this has been related to reduced PGE 2 production (Bowery and Lewis, 1973). The effects of flurbiprofen noted earlier, are consistent with this mechanism also operating in BAT. Margules et al., (1978) have suggested that fl-endorphin may be involved in the regulation of energy balance and could be responsible for the genetic obesity in some rodents. They report that pituitary levels of ACTH, corticotrophin-like intermediate-lobe peptides (CLIP) and/~-endorphin are dramatically increased in genetically obese rats and mice and that hypophysectomy or treatment with the endorphin antagonist naloxone reduces the obesity and food intake of these animals. Since the obesity of these mutants results largely from an impairment of thermogenesis (Thurlby and Trayhurn, 1979), these results infer that the endorphins may be involved in DIT. However, we have failed to detect any effect of acute injections of morphine or naloxone on metabolic rate in control and cafeteria rats (Rothwell et al., 1981c) but in the absence of a selective long-acting opiate antagonist it has been impossible to assess the effects of chronic treatment. Gale (1973) has reviewed the evidence for plasma osmolarity or Na + concentration acting as a thermal input signal for hypothalamic temperature regulation. Snellen (1972) has proposed that central osmoreceptors may detect mean body temperature and body mass from plasma Na ~ concentration, so that integration of these two parameters yields body heat content. In fact the rise in plasma osmolarity resulting from exercise (Snellen, 19721 or saline ingestion (Nielsen et al., 1971) leads to an elevation in regulated temperature whereas hyperhydration is associated with a fall in temperature (Snellen, 1972). These observations are consistent with the finding of sodium sensitive neurones in the preoptic/anterior hypothalamus and the proposal that the ratio of calcium to sodium in the hypothalamus may determine the set point for body temperature regulation (Myers and Veale, 1970; 1971). Replacing the drinking water of rats with 0.9'~o saline for 7 days or more has no effect on food intake or body weight gain but causes an enhanced thermogenic response to noradrenaline and a marked increase in BAT mass and protein content (Rothwell and Stock, unpublished data). Withdrawal of saline from adrenalectomized rats exacerbates the effects of corticoid insufficiency on thermogenesis in response to cold exposure (Maickel et al., 1967b): In view of these data it is interesting to speculate that the rise in plasma osmolarity associated with feeding may be in part responsible for the increase in body temperature and metabolic rate immediately aIter feeding and the hypertrophy of BAT accompanying chronic hyperphagia. Changes in extracellular sodium concentration do enhance noradrenaline stimulation of respiration in isolated BAT cells (Nedergaard, 1981}. However, it is unlikely that physiological changes in plasma sodium would be large enough to affect this mechanism and the above findings may therefore be due to central actions of sodium. JpI

['7_~.1

264

N.J. Roli!',~,i:i i. M. J. S]()('K and I). STRIIII 1~(;

5. C O N C L U S I O N S From this review, it is obvious that DIT is very similar to NST, and therefore much of the work on cold adapted animals is now relevant to DIT and energy balance. This similarity has proven very useful in the study of the mechanisms of DIT. which have only recently been investigated in any detail and yet our knowledge of this subject is now almost as great as NST. Noradrenaline is of major importance in the peripheral activation of thermogenesis and although many other hormones are involved, most of these seem to bc permissive in action. The factors involved in the chronic adaptation to cold and hyperphagia remain uncertain and apparently involve additional factors to the acute responses. Considerable data exist on the central pharmacology of NST and food intake but very little is known regarding DIT. It has been suggested that the ventromedial hypothalamus (VMH), which is involved in the control of food intake may also bc important for DIT. Electrical stimulation of this area causes actiwttion of lipogencsis (Shimazu and Takahashi, 1980) and thermogenesis (Perkins et al., 1981) in BAT, whereas lcsions of the VMH produce atrophy of the tissue (Seydoux et al.. 1981) and reduced thermogenesis which may be contributory to the development of obesity. These findings suggest a close relationship between the neural mechanisms of food intake control and energy expcnditure, so that like N S T , much of the data already reported on food intake could be applicable to DIT. Perhaps one of the greatest areas of ignorance in this ticld concerns the afferent pathways for energy balance regulation and DIT. In terms of NST, peripheral thermoreceptors probably provide the afferent stimulus, but it is not known whether changes in energy intake or body energy stores activate DIT, or whether the receptors arc located centrally or peripherally. In rodents and other small mammals. BAT is the major thermogenic tissue, but in larger animals the total mass and activity of brown fat is much lower, and declines rapidly with age. For example, the pig is claimed to have no brown fat, but young piglets can exhibit DIT, indicating that some alternative pathway could be involved. Infusions of noradrenaline also stimulate metabolic rate in the young pig and this is associated with dramatic increases in blood flow to white adiposc tissue in areas usually associated with brown fat. This suggests that either white adiposc tissue is rcsponsiblc for thcrmogcnesis in the pig or that brown fat cells are located in these areas but are too diffuse to identify. Similar problems exist in man, and although histological examination of cadavers has clearly demonstrated the presence of BAT in adult man (Heaton, 1972), it is impossible to assess the amount, or activity of this tissue. There is some circumstantial evidence to show that BAT can be activated in adult humans, since sympathomimetic agents which stimulate metabolic rate also cause large increases in skin temperature over areas where BAT is located (Rothwell and Stock, 1979b). Howcver, the contribution of BAT to NST or DIT in man is unknown, but it seems likely that the mechanisms of thermogenesis may be similar to those seen in other mammals. REFERENCES AFZI¢I.It:S. B. A. 11970)Brov, n adipose tissue: Its gross anatom,,, histology, and c.',tolog.~. In: Bro~n .,Idipo.w Ti.ssue, LINI)lllCrc; O. led). pp. 1 31, Elsevier. At.l:xa>,lDEr, G.. Blyyt:lq-. J. W. and GVMMII.t.R.T. 119751 Brown adipose tissue in the ncv,-born calf. J. Physiol. 244:223 234. ALeX,)N, S., NI-:uI!rGAarD. J., ()ss~l;nl)se y, |1. and ( ' a y x o y . B. (1981) Peroxisomcs in brov, n hit. In Satellite ~¢" 28 Int. ('ongr. Ph.vsiol. Sci. Pets. Szt LI ~','! S. and S/i ~.1L', M. (eds). pp. 438-485. Pergamon Press. Oxford. BI-:NN!,J L T.. G a t J , It'. A. M.. Gr!:!!y. J.. Mac!x)>,aLp. I. A. and WA! I-orb. S. (19801 l h c influence of fl-adrenoreceptor antagonists on thermoregulation during insulin-induced hyp0gl,,cacmia. J. Physiol. 208: P 26. Ba,',iFr, M., HFYSEL. H. and LH!R!:rmay. H. ~197~1 Central control of shivering and non-shivering thermogcncsis in the rat. J. Physiol. 283: 569-584. BowErY, B. and L!!w!s. G. P. (1973) Inhibition of functional dilatation and prostaglandin formation in rabbit adipose tissue by indomethacin and aspirin. Br. J. l'harmuc..17: 305314. Bray, G. A. and YorK, D. A. (1979) Hypothalamic and genetic obesity in experimental animals: an autonomic and endocrine hypothesis. Ph.!'siol. Rer. 59:719 809.

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11OI!BI!L, B. and 'I'l![rFl.l~at'M. P. 11966) Weight regulation in normal and hypothalamic h~pcrphagic rats..I. comp. Physiol. Psycho/. 61 : 180.- 193. Hemv,'lrz. FI. A. (19731 Ouabain-sensitive c o m p o n e n t of brown t~3t thcrmogenesis. Am. J. t~lzvsiol. 224:352 355. ItoRwrrz. B. A. (1979J Cellular events underlying catecholamine-induccd thcrmogenesis: cation transport in brov,'n adipocytes. Fed. Proc. 38:2170. 76. HORv,Tr:,:, B. A. and t-l,x:qis. G. E. (19761 Propranolol and p',rogen effects on shivering and non-shi',ering thcrmogenesis in rats..,|hi. J. PhyMol. 27,A): 637 &..~2. IKl!M(rro. H.. [hRoStll(iF. r . and IroH, S. (1967) ()x3,gcn consumption of brown rift in normal and hypoth,,roid mice. Jup. J. Ph.~siol. 17: 516--522. [NGR.-XM. D. L. and K.,'~('lll~)-Us[ It.KO. H. (19801 Metabolic effects of glucagon in the yotmg pig. Horm. Metah. Re.s. 12:430 433. JAXSKY. L. 11973) Non-shi',ering thermogencsis and its thcrmorcgulatory significance. Biol. Ret. ,18:85 132. J[-SSliy, K. 119801 An assessment of h u m a n regulatory nonshivermg thermogenesis..,lcta antw.~th. ~caml. 24: 138 143. JoN, C. D. 11966) Stimulation of metabolism of rat brown adipose tissue by addition of lipolytic hormones m t'itro. J. biol. Chem. 2,.11:814 821. K,~f'll:l~o-UsC'tl,KO. H. and IYC;R,xM. D. L. (19771 The effect of proprauolol of cold-induced thermogcncsis in the pig. Comp. Bio~hem. Ph).siol. 56c: 53 55. KA,~,XRI'K. R. B. and BECK, J. M. ~1980) Role of gonadal hormones in diet selection and food utilization in female rats. Phy,siol. Behar. 24: 38l 386. K^THER, II.. W.aI.TFR. E. and SIMox, B. (1978) Prostaglandins and obesity. Lamer ii: I I 1. KNI(ittr, B. L. and MYANT, N. B. (1970) A comparison between the effects of cold exposure m t'ilo and of noradrenaline m ritro on the metabolism of the brown fat of nc~ born rabbits. Biochem..I. 119: I03 II1. KLROSHt~,t,X, A.. I,)ol. K. and OH>;O, T. 11977) Effects of adrenalectomy and thyroidectomy on in riro action of glucagon on brov, n adipose tissue. J. physiol. Soc. Japan. 39:405 468. Kt:ROSHIMA. A.. Dol, K. and OH,xo, T. (1978~ Role of glucagon in metabolic ,acclimation to cold and heat. Li/e Sci. 23: 1405-.14113. Kun¢~smr,lA, A , Ko>~:
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