4

Factors Affecting Brown Adipose TissueActivity in Animals and Man

DANIEL RICQUIERGERARD MORY

Brown adipose tissue (BAT) is the only known tissue whose main functionis heat production. BAT is an important site of non-shivering thermogene­sis and perhaps of diet-induced thermogenesis. Thus. this tissue probablyplays a significant role in the regulation of energetic equilibrium in mostmammals. Before describing those factors which control BAT activity, wewill briefly describe its organization and function. The specialist reader isreferred to the great number of excellent reviews on BAT and itsmitochondria which have recently been produced (Cannon and Lindberg.1979; Nicholls. 1979; Lindberg, Cannon and Nedergaard, 1981; Neder­guard and Lindberg. 1982; Girardier, 1983; Nicholls and Locke, 1983; Can­non and Ncdergaard, 1984a and b; Himms-Hagen, 1984; Nedergaard andCannon, 1984a; Nicholls and Locke, 1984).

Between 1961 and 1965. several groups of workers showed that heat pro­duction was the principal function of BAT. This conclusion was based onstudies of three different physiological states where a mammal's bodyrequires heat production: birth, acclimation to cold, and arousal fromhibernation (reviews: Smith and Horwitz, 1969; Hull and Hardman, 1970).

PHYLOGENETIC DISTRIBUTION, BODY LOCATION ANDl\IORPHOLOGY

BAT exists in most mammals but has never been identified in non­mammals. even in homoiothermic animals such as birds. In the human spe­cies, BAT has been well characterized in the newborn (reviews: Hull andHardman, 1970; Cannon and Johansson, 1980; Lean and James. 1983).Although islets or pieces of BAT have been observed in human adults(Heaton, 1972; Tanuma et ai, 1976; Hassi, 1977; Huttunen, Hirvonen andKinnula, 1981), BAT in the human adult has not been quantified, and thequestion of its physiological importance is presently a subject of con­troversy (see Astrup et al, 1984).

BAT can account for 0.5 to 5 per cent of the body weight of most rnam-

Clinics ill Endocrinology and Metabolism-Vol. 13. No.3. November 1984 501

502 DANIEL RICQUIER AND GERARD MORY

mals . This tissue is generally more abundant in interscapular, cervical andthoracic regions in small mammals such as rodents and lagomorphs and inthe perirenal region of larger species such as the lamb (review: Afzelius ,1970).

BAT is composed of brown adipocytes which have characteristic fea­tures (Figure 1), with many small multilocular triglyceride droplets and anexceptionally high content of mitochondria which are well filled with cris­tae. BAT is also characterized by numerous blood capillaries and by a richnetwork of sympathetic nerves . These sympathetic fibres innervate arterialblood vessels , and they also directly innervate brown adipocytes throughabundant synaptic varicosities which run between the adipocyte cells. Thetransmitter released by these varicosities is noradrenaline (reviews: Cottle,1970; Schonbaum, Steiner and Sellers, 1970; Barnard, Mory and Nechad,1980). Several authors have proposed that sympathetic relays exist insideBAT, but this has been repudiated by recent investigations. Further, noother aminergic fibres, such as serotoninergic or dopaminergic fibres, havebeen found in BAT (review: Mory, Combes-George and Nechad, 1983).

THE MECHANISMS OF THERMOGENESIS IN THE BROWNADIPOCYTE

It is now well established that thermogenesis in the brown fat cell resultsmainly from a physiological uncoupling of oxidative phosphorylation inmitochondria. Elevated amounts of free fatty acids can be generated in thecell through lipolysis, fatty acid synthesis and importation of fatty acids.These free fatty acids are quickly oxidized inside mitochondria. In contrastto most mitochondria, where respiration and AOP phosphorylation areobligatorily linked, free energy from the oxidation of substrates is directlydissipated as heat. This uncoupling mechanism involves a specific regulatedtransport of protons across the inner membrane (reviews: Nicholls andLocke , 1983, 1984). The proton pathway is specific to brown fat mitochon­dria and is inhibited by nucleotides (AOP, ATP, GOP and GTP) and acti ­vated by free fatty acids (reviews: Nicholls and Locke, 1983, 1984). Themolecular basis for this unique H + conductance pathway is a specific pro­tein (M, 32 (00), named uncoupling protein or thermogenin, located in theinner mitochondrial membrane. Before its identification as the proteinresponsible for the uncoupling of mitochondria (Heaton et ai, 1978), thiscomponent had been shown to be present in large amounts in the mem­branes of mitochondria from active brown fat and in smaller amounts inmitochondria from weakly active BAT (Ricquier and Kader, 1976). Manystudies subsequently led to the conclusion that the total amount of uncou­pling protein in BAT determines the capacity of the tissue for thermogene­sis (reviews: Nicholls and Locke, 1984; Cannon and Nedergaard, 1984b).Thus, factors which are able to modulate the activity and the amount of theuncoupling protein are factors which control the activity of BAT.

When thermogenesis in BAT is activated at birth, during acclimation tocold, and during arousal from hibernation the heat produced by BAT rep-

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Figure 1. Electron micrographs of interscapular brown adipose tissue taken from a cold-acclimated rat.Courtesy of Dr M. Nechad. Left (la): x 2000. Right (lb): x 30000. L = lipid droplets: N = nucleus:M = mitochondria; G = glycogen: C = capillary.

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504 DANIEL RICQUIER AND GERARD MaRY

resents a large part (30 to 80 per cent) of total non-shivering thermogenesis(Hull and Hardman, 1970; Foster and Frydrnan, 1979; Nedergaard andCannon, 1984b). In addition to its considerable role in non-shivering ther­mogenesis (at least in rodents: Foster and Frydman , 1979), recent work hasimplicated BAT in diet-induced thermogenesis and regulation of bodyweight (Rothwell and Stock, 1979) (see Chapters 1 and 2 of this volume).

NEURAL FACTORS

The factors which control non-shivering thermogenesis and diet-inducedthermogenesis in BAT are probably the same factors and can be dividedinto neural and hormonal factors. Non-shivering thermogenesis is essen­tially controlled by sympathetic nerves and noradrenaline (review: Jansky,1973), and BAT is a major site of non-shivering thermogenesis (Foster andFrydman, 1979). Thus, sympathetic neural factors could control BATactivity.

Noradrenaline released by the sympathetic neural afferents in BAT isbound to receptors on the surface of brown adipocytes. Three types ofcatecholamine receptors have been detected in brown adipocytes ofrodents. Classed according to number and physiological function, the mostimportant are B-adrenoreceptors which are associ ated with adenylatecyclase activity and cAMP production. They exhibit an affinity pattern foragonists, characteristic of the B.-subtype: isoprenaline (isoproterenol) »noradrenaline> adrenaline » phenylephrine (reviews: Girardier, 1983;Cannon and Nedergaard, 1984a) . However, Arch et al (1984) have synthe­sized and tested a new B-agon ist which specifically stimulates B-receptors ofBAT, and they concluded that these receptors are not true B.-receptors.

Brown adipocytes of rodents also contain aI-receptors, of which the pat­tern of affinity for a-antagonists is prazosin > phentolamine » yohim­bine (Mohell, Nedergaard and Cannon, 1983; Mohell, Svartengren andCannon , 1983). As for a.-receptors in other cells, calcium and phospha­tidylinositol are involved in the activation of aI-receptors of brown fat cells(Garcia-Sainz, Hasler and Fain, 1980; Mohell, Wallace and Fain, 1984;review: Nedergaard et al, 1984).

Rat brown adipocytes also possess az-adrenoreceptors, the stimulationof which inhibits B-effects (Sundin and Fain, 1983). Such receptors werenot found in hamster BAT (McMahon and Schimmel, 1982). These threetypes of receptors (B., a. and a2) were found in lamb brown adipocytes(Fain et al, 1984). The catecholamine receptors of BAT vasculature havenot been studied.

Although noradrenaline, which is the natural mediator of thermogenesisin BAT, can interact with both a-adrenergic and B-adrcnergic receptors,most heat produced by brown fat cells results from interaction with ~I­

receptors (Mohell, Nedergaard and Cannon, 1983; review: Cannon andNedergaard,1984b).

BROWN ADIPOSE TISSUE ACfIVITY 505

Sympathetic Activation of Brown Adipose Tissue

Acute exposure to cold induces a rapid increase in noradrenaline release inBAT (Cottle, 1970; Young et aI, 1982). This sympathetic activation per­sists if exposure to cold is chronic (Cottle, 1970; Kennedy, Hammond andHamolsky, 1977). Such an elevated release of noradrenaline induces only amoderate 'down-regulation' of the number of B-receptors, but there is alsoreduced coupling between B-receptors and cyclase (Svartengren, Svobodaand Cannon, 1982).

Dietary intake also modulates the release of noradrenaline in BAT. A48-hour fast markedly decreases sympathetic activity in rat BAT (Young etaI, 1982), while chronic overfeeding using the 'cafeteria diet' enhancesnoradrenaline release in BAT (Young et aI, 1982). However, short-term oracute sucrose-overfeeding affects this release only slightly and contrastswith that observed during acute exposure to cold (Young et aI, 1982): seeChapter 3.

Acute cold exposure

The response of BAT to acute cold exposure is mainly metabolic. The lipidstores are rapidly mobilized to supply fuel to the mitochondrial machinerywhich produces heat through uncoupling of oxidative phosphorylation.Within one minute of the start of lipolysis in the brown adipocyte , the cellcan increase its respiration rate 10 to 40 times. Free fatty acids are not onlysubstrates for mitochondrial respiration but also immediately increase pro­ton conductance of the inner mitochondrial membrane and hence uncouplerespiration (Rial, Poustie and Nicholls, 1983; review: Nicholls and Locke,1984). A marked increase in the ability of GDP to bind to isolated BATmitochondria also occurs within the first hour of exposure of rats to cold(Desautels, Zaror-Behrens and Himrns-Hagen, 1978). GDP binds to theuncoupling protein (Heaton et aI, 1978), and the binding is considered toreflect the activity of the proton conductance pathway of BAT mitochon­dria. During a short exposure to cold, there is also a rapid increase inlipoprotein lipase activity in BAT which allows the tissue to oxidize circu­lating lipids (Radomski and Orme, 1971; Cameheim, Nedergaard andCannon, 1984). Moreover, the metabolic activation of brown fat cells isaccompanied by a large increase in blood flow through the tissue(Kuroshima, Konno and Itoh, 1967; Foster and Frydrnan, 1979).

All these events are undoubtedly mediated by the activation of thesympathetic nerves in BAT and the subsequent release of noradrenaline.It has long been known that surgical denervation of BAT prevents lipidmobilization in the tissue during cold exposure (Hausberger, 1934; review:Girardier, 1983). Oxidative metabolism of isolated brown adipocytes canbe directly increased in vitro by adding noradrenaline and almost totallysuppressed by adding B-blockers such as propranolol (reviews: Lindberg,Bieber and Houstek, 1976; Nedergaard and Lindberg, 1982; Cannon andNedergaard, 1984a). The catabolic cascade starts with the binding ofnoradrenaline to B-receptors and continues with an increase in cAMP con-

506 DANIEL RICQUIER AND GERARD MaRY

centration and activation of hormone-sensitive lipase. According to recentdata, 80 per cent of the increase in oxygen consumption following noradre­naline binding to brown fat cells results from stimulation of the B-receptoradenyl ate cyclase system and 20 per cent corresponds to an a-receptor­mediated effect (Mohell, Nedergaard and Cannon, 1983). The rapidincrease in GDP binding to isolated mitochondria of rats during acute coldexposure can be induced by a l-hour intravenous infusion of noradrenaline(Desautels and Himms-Hagen, 1979), and this effect can be largelyimpaired by pretreating the animals with propranolol (Mory et aI, 1984b).The enhancement of lipoprotein lipase activity during short cold exposurecan be reproduced by injecting isoprenaline or noradrenaline and isblocked by propranolol (Radomski and Horme, 1971; Carneheim, Neder­gaard and Cannon, 1984). The increase in blood-flow which is characteris­tic of the response of BAT to cold is also a consequence of stimulation ofthe tissue by noradrenaline (Hull and Hardman, 1970; Foster andDepocas, 1980). Acute cold exposure causes mobilization of serotoninfrom BAT mast cells (Mory, Combes-George and Nechad, 1983) which inturn can stimulate noradrenaline release from nerve endings (Steiner andEvans, 1976) and can thus amplify sympathetic activation.

Chronic cold exposure

When animals are chronically exposed to cold, the metabolic activation ofBAT persists and lipolysis, lipogenesis, oxygen consumption and lipopro­tein lipase activity remain elevated (review: Nicholls and Locke, 1984).With prolonged cold exposure in adult animals, BAT shows further adap­tive changes which are the opposite of the normal involution expected inthis tissue after the first month of life in most non-hibernating rodents. Thisadaptive response can be detected within three to five hours of exposure tocold and reaches a maximum after several weeks of cold exposure (Table1). Cold acclimation of rats also leads to increased blood flow throughBAT (Kuroshima, Konno and Itoh, 1967, Foster and Frydman, 1979). Theorgan also proliferates with endothelial precursor celis maturing and lead­ing to a doubling or tripling of the organ's DNA content (review: Barnard,Mory and Nechad, 1980). Mitochondrial mass also increases markedly onan organ and cell basis; the proportion of the 32 OOO-M r mitochondrialuncoupling protein is also strikingly increased (reviews: Nicholls andLocke, 1984; Nedergaard and Cannon, 1984a). The selective enrichment inuncoupling protein can be detected within 3 h of cold exposure (Mory etaI, 1984b). These events are probably explained by the observation that thelevel of mRNA encoding the uncoupling protein increases during pro­longed exposure to cold (Ricquier et ai, 1983b; Bouillaud et aI, 1984).Moreover, numerous enzymatic activities, such as cytochrome c oxidaseactivity, are enhanced and the fatty acid composition of phospholipids isaltered (review: Barnard, Mory and Nechad, 1980). All the modificationsobserved during prolonged cold exposure allow a striking increase in theheat production capacity of BAT.

Several observations clearly indicate that the integrity of BAT innerva-

Table 1. The trophic response of brownadipose tissue of rats acclimated to coldand itscontrol by the sympathetic innervation

ChemicalChronic cold sympathectomyand Daily injection Continuous delivery Phacochromocytoma

exposure" cold exposure" of catccholamines" of noradrenaline" tumour (PC 12cells)"

Weight of BAT + 0 + + +DNA content + 0 + + +Protein concentration + 0 + + +Phospholipidconcentration + 0 0 n.d, +Unsaturation degree of phospholipids + 0 0 n.d, +Mitochondrialprotein concentration + 0 n.d. + +Cytochrome c oxidase activityper cell + 0 + + 0Uncouplingprotein concentration in mitochondria + 0 0 + +GDP binding to isolated mitochondria + 0 0 + +

+ = Large increase.o= No or weak increase.n.d. =Not determined.• Data from Barnard, Mory and Nechad (1980)and Mory et al (1982).b Data from Desautels and Himms-Hagen (1979)and Mory, Ricquier and Hernon (1980).C Data from Mory et al (1984a).d Data from Ricquier ct al (1983a).

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508 DANIEL RICQUIER AND GERARD I\IORY

tion is necessary for development of the tissue. After 5 h cold exposure,normal cell proliferation does not occur in surgically denervated BAT(Hunt and Hunt, 1967), and the increase in the uncoupling protein concen­tration is inhibited by pretreatment of animals with propranolol (Mory etai, 1984b). Likewise, the major features of the trophic response of BAT incold-acclimated animals (increases in DNA, total and mitochondrial pro­tein, cytochrome c oxidase activity and uncoupling protein, alterations ofphospholipids) are suppressed or largely impaired in rats chemically sym­pathectomized using guanethidine treatment (Mory et ai, 1982). The roleof innervation has also been studied using BAT grafts (Nechad and Olson,1983).

Daily injections of noradrenaline or of isoprenaline in rats for severalweeks can induce growth of BAT, including hyperplasia and increasesin protein content and cytochrome c oxidase activity (Leblanc andVillemaire, 1970; Heick et al, 1973; Desautels and Himms-Hagen, 1979;Mory, Ricquier and Hernon, 1980). However, these rats did not exhibit thecharacteristic increase in uncoupling protein observed during cold exposure(Desautels and Himms-Hagen, 1979; Mory, Ricquier and Hernon, 1980).This observation implied that noradrenaline and sympathetic innervationwere necessary but not sufficient to induce the full adaptive response ofBAT. However, recent experiments with continuous infusion (rather thansingle daily injections) of noradrenaline, using implanted mini-osmoticpumps, showed that BAT of noradrenaline-infused rats developed in a waysimilar to that observed in cold-acclimated rats (Mory et ai, 1984a). Theproportion of mitochondrial uncoupling protein in these experimentsincreased as much as in those experiments in which animals were exposedto prolonged cold. The effect was observed only when the pump deliveringnoradrenaline was placed near interscapular BAT, and not when the pumpwas implanted 6 to 9 em away from the tissue (Mory et ai, 1984, unpub­lished data). These data favour the proposition that noradrenaline releasedby nerve endings in BAT is the agent responsible for the adaptive trophicresponse of BAT. This conclusion is in excellent agreement with theobservations made by Arch et al (1984) and by Young, Wilson and Arch(1984). These authors have shown development of BAT and a specificincrease in the uncoupling protein in rats chronically treated with long­acting ~-adrenoreceptor agonists such as fenoterol or by a new compound(from Beecham Laboratories) which has a strong affinity for the ~­

adrenoreceptors of BAT.

Phaeochromoeytoma in Animals and Humans

The development of BAT, and sometimes of BAT pseudotumours, hasbeen described in humans and animals bearing phaeochromocytomas­tumours which secrete large amounts of catecholamines (reviews: Feyrter,1973; Girardier, 1983). The implantation of phaeochrornocytoma tumours(PC 12 cell line) in rats induces the development of BAT and stimulates thesynthesis of the uncoupling protein (Ricquier et ai, 1983a; Bouillaud et ai,1984). An ultrastructural and biochemical study of perirenal fat from

BROWN ADIPOSE TISSUE ACTIVITY 509

humans bearing phaeochromocytoma tumour has revealed that this tissuewas typical of BAT, with mitochondria behaving biochemically like BATmitochondria (Ricquier, Nechad and Mory, 1982; Bouillaud, Combes­George and Ricquier, 1983). Conversely, perirenal fat in patients bearingno phaeochromocytoma resembles typical unilocular white adipose tissue.Interestingly, it has been reported that brown fat pseudotumours can beconfused with the tumour itself in patients with phaeochromocytoma(review: Girardier, 1983). Thus, sympathetic factors released by tumorouschromaffin cells (probably catecholamines) are able to stimulate BATdevelopment and to induce maturation of precursor cells and/or 'dedif­ferentiation' of unilocular fat cells. Furthermore, the apparent transforma­tion of white fat cells into brown adipocytes has been described in severechronic hypoxaemia which induces sympathetic activation in man (Teplitzet al, 1974). The major effects induced by catecholamine infusions or in thepresence of phaeochromocytoma are indicated in Table 1.

The Role of Neural Factors in the Response to Diet

Rats overfed a cafeteria diet have more BAT than animals fed a stock diet(Rothwell and Stock, 1979; Chapter 1). This growth of BAT is due tohyperplasia (Himms-Hagen, Triandafillou and Gwilliam, 1981; Buko­wiecki et al, 1982), and GOP binding to isolated BAT mitochondriaincreases (Brooks et ai, 1980; Himms-Hagen, Triandafillou and Gwilliam,1981). Moreover, blood-flow through BAT is largely increased (Rothwelland Stock, 1981a). Thus, chronic overeating and chronic cold exposureinduce almost similar modifications in BAT, leading to an enhancement ofthe thermogenic capacity of the tissue (see Chapter 1). Furthermore, asingle meal has been reported to activate BAT metabolism (Glick, Teagueand Bray, 1981). However, an increase in the proportion of uncouplingprotein has not been observed so far (Himms-Hagen, Triandafillou andGwilliam,1981).

As in the case of cold exposure, a great number of observations haveshown that sympathetic innervation of BAT and released noradrenalineplay key roles in the development of BAT during chronic overfeeding(review: Stock and Rothwell, 1981). Chronic cafeteria feeding increasesnoradrenaline release in BAT of rats (Young et al, 1982), and the trophicresponse of BAT in rats overfed with sucrose is impaired by chemicalsympathectomy (Sundin and Nechad, 1983). Conversely, BAT of obeseanimals has a low thermogenic capacity (see Chapter 2 of this issue)which is associated with a decrease in noradrenaline turnover in the tissue(Knehans and Romsos, 1982; Levin, Triscari and Sullivan, 1983) andwith a chronic lack of sympathetic activation of BAT (Seydoux et al, 1981).

Conclusion: Neural Factors

The evidence of the interaction between BAT and its sympathetic innerva­tion clearly shows that the neural distribution within this tissue plays anessential role in the control of tissue activity. Although we cannot exclude

510 DANIEL RICQUIER AND GERARD MORY

the possibility that a neurotransmitter distinct from noradrenaline mod­ulates BAT activity, we conclude that noradrenaline released by nerveendings in BAT is the major mediator of thermogenesis in BAT. Furth­ermore, it is known that the activity of BAT sympathetic nerves is control­led by two hypothalamic areas: the preoptic area (POAH) and the ven­tromedial area (VMH). The former area is sensitive to temperature andthe latter area to feeding conditions (review: Girardier, 1983; Chapter 5).

Although true activation of BAT by cold is well described in animals(e.g. rodents), this phenomenon has not yet been clearly demonstrated inadult man. Exposure to cold results in a general stimulation of sympatheticactivity, so the effect on the sympathetic innervation of BAT during coldexposure and acclimation to cold may not be specific.

The observations on phaeochromocytoma in man and animals suggestthat chronic general catecholaminergic stimulation can lead to prolifera­tion of BAT, and these tumours are clinically, of course, associated withweight loss. This weight loss may therefore relate to the enhanced activityof the proliferated mass of brown adipose tissue. Studies on the BAT ofelderly subjects dying of hypothermia show lipid depletion of their brownadipose cells, a finding mimicked in newborn babies with hypothermia(Aherne and Hull, 1966). These observations, together with the autopsyfindings on adult Finns who were chronically exposed to cold and showedgreater quantities of BAT (Huttunen, Hirvonen and Kinnula, 1981), sug­gest that sympathetic activation of BAT does occur in man and seems to bemediated by catecholamines. Whether enhanced BAT activity contributessignificantly to maintaining body temperature in the cold-exposed adult isdoubtful, although it has been suggested as the principal organ involved inthe substantial increases in metabolic rate accompanying non-shiveringthermogenesis in the newborn (Hey, 1975).

HORMONAL FACTORS

Significant hormonal changes also occur when mammals exhibit increasednon-shivering thermogenesis or increased diet-induced thermogenesis andcould therefore be involved in the modulation of BAT activity.

Thyroid Homones

Thyroid hormones stimulate thermogenesis and are necessary for survivalin cold. However, despite the finding of an elevated level of T) in cold­acclimated rats (review: Himms-Hagen, 1983a), it is now clear that T) doesnot mediate the effect on BAT.

Authors have compared BAT from hyperthyroid and from cold­acclimated animals. The changes induced in BAT by cold exposure werenot mimicked in animals given injections of T4 (Leblanc and Villemaire,1970; Heick et al, 1973; Ricquier, Mory and Hernon, 1975, 1976, 1978;Harri, 1978), and the enlargement of BAT in hyperthyroid rats is due notto an increase in protein content but to an increase in triglyceride content

BROWN ADIPOSE TISSUE ACfIVITY 511

(Lachance, 1953; Heick et ai, 1973; Ricquier, Mory and Hernon, 1976;Triandafillou, Gwilliam and Himrns-Hagen, 1982). Recently, it has alsobeen reported that thyroxine injections in rats decrease the level ofmitochondrial uncoupling protein, measured by GDP binding to mitochon­dria (Sundin, 1981; Triandafillou, Gwilliam and Himms-Hagen, 1982), andprevent the normal cold-induced changes occurring in BAT (Ricquier,Mory and Hernon, 1976, 1978).

Other studies have been carried out in hypothyroid animals . Such anim­als have a BAT composition similar to that found in the cold-acclimatedanimal-e .g., increases in wet weight, DNA content, mitochondrial pro­tein, cytochrome c oxidase activity (Mory et ai , 1981). However, BATfrom hypothyroid animals is refractory in its reaction to noradrenaline asshown by (1) a poor lipolytic response to added noradrenaline (review:Hernon, Ricquier and Mory, 1976) or to cold stress (Mory et ai, 1981); (2)a sharply decreased metabolic response to nerve stimulation (Seydoux,Giacobino and Girardier, 1982); and (3) no increase in blood-flow duringchronic cold exposure (Kuroshima, Konno and Itoh, 1967). Moreover,although the amount of mitochondrial uncoupling protein per cell was notincreased in hypothyroid rats, the total amount in the organ was doubled(Mory et al, 1981; Triandafillou, Gwilliam and Himms-Hagen, 1982). Sel­lers, Flattery and Steiner (1974) had previously observed that hypothyroidrats provided with the minimum dose of T, for survival in cold were able todevelop their BAT during prolonged cold exposure.

It is noteworthy that, in 1850, Curling described 'symmetrical swellingsof fat tissue at the side of the neck' in human babies with cretinism. Later itwas recognized that this tissue was BAT (Shattock, 1909). The develop­ment of BAT in young cretins is due mainly to accumulation of fat becauseof inhibited lipolysis and a high lipoprotein lipase activity (Hernon, Ric­quier and Mory, 1975, 1976).

This complex involvement of the thyroid (also noted in Chapter 8) maybe simply explained as follows: in comparison with euthyroid animals,hyperthyroid animals have increased metabolism with a rise in bodytemperature and therefore a reduced drive for BAT thermogenesis fromthe hypothalamus via the sympathetic nervous system. The oppositemechanism could occur in the hypothyroid animal. Nevertheless,hypothyroidism does not completely mimic the effects of cold exposure,since noradrenaline sensitivity of BAT is abnormal.

The role of thyroid hormones in BAT may therefore be restricted to apermissive role (Triandafillou, Gwilliam and Himrns-Hagen, 1982;Hernon, Ricquier and Mory, 1974; Sellers, Flattery and Steiner, 1974).These hormones may neither stimulate the activity of BAT directly normediate the cold-induced development of the thermogenic capacity of thetissue. We can unequivocally conclude that BAT is not a target of thyroid­stimulated thermogenesis.

Pancreatic Hormones

It has been reported that glucagon secretion is increased during cold expo­sure and that this hormone is able to directly stimulate lipolysis in BAT

512 DANIEL RICQUIER AND GERARD MaRY

(review: Kuroshirna et al, 1984). Glucagon has a marked calorigenic actionwhen it is added to isolated brown adipocytcs but such an effect is obtainedwith a concentration of glucagon which is 3000 times the glucagonaemiameasured by the same authors . Thus, the hypothesis of a physiological rolefor glucagon in the induction of BAT thermogenesis is doubtful.

Insulin has obvious metabolic effects on isolated brown adipocytes orfragments of tissue. These effects are in many ways similar to thoseobserved in white adipose tissue (reviews: Nedergaard and Lindberg, 1982;McCormack, 1982). These effects include stimulation of glucose uptake,glucose oxidation and oxygen consumption, and stimulation of fatty acidsynthesis via activation of pyruvate dehydrogenase and acetyl-CoA car­boxylase. There is a decrease in adrenaline-stimulated glycerol and freefatty acid release, and a smaller rise in cyclic AMP induced by addednoradrenaline.

BAT is a major site of lipogenesis in the cold-adapted rat (McCormackand Denton, 1977; Trayhurn, 1979; review: McCormnck, 1982). Glucose isthe predominant substrate for fatty acid synthesis in BAT (review: McCor­mack, 1982) and this process is stimulated by insulin (McCormack andDenton, 1977; Agius and Williamson, 1980; Agius et al, 1981). It is alsoknown that glycolysis in the brown fat cell is necessary for thermogenesis inorder to increase the level of citric acid cycle intermediates (Nedergaardand Lindberg, 1982). McCormack (1982) has proposed that, underappropriate circumstances, glucose could be a substrate for oxidation inBAT, and that 'the function of insulin could be seen as a switch mechanismwhereby the fat fuel present in the tissue for thermogenesis can be con­served and augmented when glucose, as an alternative fuel is available inthe bloodstream'. Increased lipogenesis in BAT does not necessarily meanthat there is increased thermogenesis in the tissue.

An increase in BAT temperature is obtained in response to electricalstimulation of the ventromedial hypothalamic nucleus (Perkins et al,1981). Such stimulation of the YMH enhances lipogenesis preferentially inBAT, in normal and in diabetic animals (Shimazu and Takahashi, 1980).This effect is probably obtained via activation of the sympathetic innerva­tion of BAT. Thus, although insulin undoubtedly stimulates lipogenesis inisolated brown fat cells, such a process can also be stimulated by othermediators. Perhaps the effects of insulin on BAT in vivo are indirect anddue to its action on glucose receptors in the YMH zone. This hypothesisallows for the reduction in BAT diet-induced thermogenesis in diabeticanimals (Goodbody and Trayhurn, 1981; Seydoux et al, 1983, 1984) and inrats with hypothalamic obesity (Seydoux et al, 1981, 1982; Himms-Hagen,1983b).

Seydoux et al (1983, 1984) have studied BAT of streptozotocin-diabeticrats and of rats chronically infused with insulin. The data obtained indicatethat insulin increases BAT mass, metabolic capacity of BAT, fatty acid B­oxidation, and the concentration of uncoupling protein in mitochondria.At present, it is not known whether or not all or some of these insulineffects are due to secondary increased sympathetic activity in BAT, but ahigh capacity of BAT for non-shivering thermogenesis requires insulin. It

BROWN ADIPOSE TISSUE ACTIVITY 513

has also been suggested that insulin is necessary for the diet-induced ther­mogenic response in BAT (Rothwell and Stock, 1981b). However, thetrophic response of BAT to cafeteria feeding was not dependent on insulin,although the noradrenaline-mediated thermogenic response of BAT inthese animals required insulin (Rothwell and Stock, 1981b). The under­standing of the role of insulin in BAT of cafeteria-fed animals is compli­cated by the fact that authors have reported opposite responses of insuli­naernia in these animals (Rothwell and Stock, 1981b; Cunningham et al,1983). On the other hand, the BAT in obese infants of diabetic mothers(Aherne and Hull, 1966) has more lipid than normal so the higher insulinoutput of these infants may well have affected BAT metabolism.

In conclusion, insulin stimulates glucose metabolism, fatty acid synthesisand thermogenesis in BAT. These effects of insulin are probably induceddirectly and indirectly via activation of the YMH area. Recently, a markedincrease in the glucagon and insulin content of BAT has been found in thecold-acclimated rat , but the significance of these unconfirmed findings isunclear (Kuroshima et al, 1984).

Adrenal Hormones

Although adrenaline is able to stimulate thermogenesis in brown fat cells,it is unlikely that adrenaline released by adrenals plays a role in activationof BAT since the circulating level of adrenaline is normally too low for thispurpose (Girardier, 1983). The effects of phaeochromocytomas havealready been discussed.

The level of uncoupling protein (as estimated by GDP binding to iso­lated mitochondria) is lower in obese Zucker rats than in lean animals, butthis low level can be restored to normal if the fatty rats are adrenalecto­mized (Holt and York, 1982; Holt, York and Fitzsimons, 1983). Brown fatof Zucker rats is reactivated following adrenalectomy (Marchington et al,1983), and brown fat is suppressed in mice treated with corticosterone(Galpin et al, 1983). In clinical pathology, it has been claimed that patientswith Addison's disease show a growth in BAT (Afzelius , 1970). It is recog­nized that addisonian patients have a low rate of production of adrenocor­ticoid hormones, so these may normally exert a suppressor effect on BATin vivo in humans as well as in experimental animals. The basis for theinhibitoryeffects of glucocorticosteroids and the stimulatory effects ofadrenalectomy have yet to be explained. Speculatively, it can be proposedthat adrenalectomy triggers sympathetic innervation of BAT.

Pituitary Hormones

Repeated injections of rats with ACfH (Laury and Portet , 1980; Harri,1981) or acclimation of hypophysectomized animals to cold (Fellenz et al,1982; Goubern ct ai, 1984; Laury et al, 1984) has clearly demonstrated thatnone of the pituitary hormones is directly involved in the development ofthe thermogenic capacity of BAT. Interestingly, it has been observed thathypophysectomized rats kept at 28°C exhibited several alterations in their

514 DANIEL RICQUIER AND GERARD MORY

BAT which are characteristic of the cold-acclimated state (Laury et ai,1984). This may reflect the lack of inhibitory effects of T4 and glucorcorti­coids.

Other Hormones

The stimulatory effects of serotonin on brown fat (Mory and Ricquier,1981) are due to the secondary release of noradrenaline (see above). Mela­tonin is also able to stimulate the growth of BAT in hibernating species(Heldmaier and Hoffman, 1974; Sinnamon and Pivorun, 1981). Neverthe­less, the intact pineal is not a prerequisite for the cold-induced increase inBAT occurring in the cold-acclimated rat (Kott and Horwitz, 1983).

CONCLUSION AND PERSPECTIVES

We may conclude that the action of thyroid hormones can probably be res­tricted to a permissive role. Large doses of thyroxine or of glucocorticoidshave inhibitory effects on BAT activity. Among hormones, insulin seemsto be the only one which is able to stimulate (directly and/or indirectly) theactivity of BAT.

Factors which affect the activity of BAT regulate brown adipocyte prolif­eration and differentiation, the development of mitochondria, and thesynthesis of the uncoupling protein, and modulate the availability ofoxidizable substrates. We conclude that noradrenaline released by BATinnervation is the main factor controlling heat generation and developmentof the tissue. Moreover, BAT is centrally controlled by the hypothalamus,and while insulin activates BAT its role is of lesser importance.

Both in vivo and in vitro experiments emphasized the role of noradrena­line in BAT activation. Cultures of brown adipocytes have been recentlydeveloped (Nechad, 1983; Sugihara et ai, 1983; Forest et ai, 1984) and canbe used in the future to study the direct role of neurotransmitters and hor­mones in BAT development.

Most studies have been carried out in rodents rather than in man. Inorder to deal with the question of a possible role of BAT in the regulationof thermogenesis and body weight in man, it is necessary to developmethods for the unambiguous characterization of brown adipocytes inhealthy adult man. The very high thermogenic capacity of BAT is recog­nized, as is the identification of functional 'BAT in the human neonate andin adult patients. Throughout this chapter, clinical examples have beenincluded which demonstrate the involvement of BAT in the conditions ofhypothyroidism, cold exposure, Addison's disease, and phaeochromocy­toma. The role of these changes in determining associated changes inenergy balance is, of course, unknown, but they may playa part. It doesnot seem unreasonable to think that, in the foreseeable future, there willbe appropriate pharmacological treatments of some obese patients toinduce BAT development and contribute to weight loss.

BROWN ADIPOSE TISSUE ACTIVITY

ACKI'OWLEDGEMENTS

515

We wish to thank Dr Myriam Nechad for the illustration (Figure 1) and Suzann McKay for theexcellent reading of the manuscript. We also thank the following colleagues for having sent usreprints and preprints of their work : Drs J. Arch, E. Ashwell, A. Astrup, B. Cannon, Z .Glick. J. Himrns-Hagen, M. C. Laury, J. McCormack. J. Nedergaard, D. Nicholls, J.Seydoux and P. Trayhurn. We appreci ated the secretarial assistance of Marlene Darde.

Experiments of the authors were financially supported by grants from the Centre Nationalde la Recherche Scientifique.

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