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The Wenner-Gren Institute, The Arrhenius Laboratories F3, Stockholm University, Stockholm, Sweden
ABSTRACT I. A MAMMALIAN PREROGATIVE: BROWN ADIPOSE TISSUE II. NOREPINEPHRINE CONTROLS THE THERMOGENIC PROCESS A. Norepinephrine Signaling Through {beta}3-Receptors Leads to Thermogenesis 1. {beta}3-Adrenoceptors in mature brown adipocytes 2. {beta}3-Adrenoceptors do not possess properties essential for brown adipose tissue function 3. Only Gs proteins couple to thermogenesis 4. Adenylyl cyclase, cAMP, and protein kinase A mediate the thermogenic signal 5. Protein kinase A-phosphorylated proteins B. Thermogenesis Is Due to Activation of UCP1 Through Lipolysis 1. Stimulation of lipolysis stimulates thermogenesis 2. Fatty acids are the thermogenic substrates 3. The uncoupling protein UCP1 C. The {alpha}2-Adrenergic Pathway Inhibits Thermogenesis D. The {alpha}1-Adrenergic Pathway and the Cell Membrane Events III. THE LIFE OF THE BROWN ADIPOCYTE IS UNDER ADRENERGIC CONTROL A. In Brown Preadipocytes, Norepinephrine Promotes Proliferation B. In Mature Brown Adipocytes, Norepinephrine Promotes Differentiation C. Norepinephrine Directly Regulates the Expression of the UCP1 Gene D. Norepinephrine Is an Apoptosis Inhibitor in Brown Adipocytes IV. HOW SIGNIFICANT IS BROWN ADIPOSE TISSUE? A. Parameters of Activation and Recruitment 1. Parameters of activation 2. Parameters of recruitment 3. Is recruitment the effect of chronic activation? B. How to Establish Brown Adipose Tissue Involvement V. THERMOREGULATORY THERMOGENESIS A. In Acute Cold, Thermogenesis Results From Shivering B. Classical Nonshivering Thermogenesis Is Entirely Brown Fat Dependent C. Cold Acclimation-Recruited, Norepinephrine-Induced Thermogenesis Is Entirely Brown Fat Dependent 1. UCP1-independent thermogenesis 2. UCP1-dependent (brown fat-derived) norepinephrine-induced thermogenesis 3. Epinephrine-induced thermogenesis 4. Glucagon-induced thermogenesis: does it exist? D. Postnatal Thermogenesis 1. Altricial newborns recruit brown adipose tissue after birth 2. Immature newborns recruit brown adipose tissue with a delay 3. Precocial newborns have recruited brown adipose tissue at birth E. Fever, Hyperpyrexia, and Anapyrexia (Stress, Anesthesia, Thyroid Thermogenesis, Exercise) 1. Classical experimental fevers 2. Stress fevers: do they represent hyperpyrexia or hyperthermia? 3. Anesthestic hypothermia 4. Thyroid thermogenesis 5. Exercise counteracts brown adipose tissue thermogenesis F. Hibernation and Arousal 1. Prehibernation fattening 2. Entry into hibernation 3. During deep hibernation 4. Arousal depends on brown adipose tissue thermogenesis 5. Daily torpor 5. Photoperiod: how does it lead to recruitment? G. The Central Regulation of Thermoregulatory Thermogenesis and the Innervation of Brown Adipose Tissue 1. The temperature control area in the preoptic chiasma/anterior hypothalamic nuclei 2. The ventromedial hypothalamic nucleus 3. The inhibitory center in the lower midbrain 4. Raphe nuclei 5. Inferior olivary nucleus 6. The intermediolateral neurons 7. The sympathetic chain (stellate ganglia) VI. METABOLOREGULATORY THERMOGENESIS A. The Acute Thermal Effects of Eating 1. Effects of a single meal 2. Fasting, food restriction, starvation: decreased activity of brown adipose tissue 3. Basal metabolic rate: a regulated entity? B. Recruiting Diets (Obesity, Leptin, Cachexia) 1. Are recruiting diets protein-diluting diets? 2. Is ''diet-induced thermogenesis'' really obesity-induced thermogenesis? 3. Are diet-adapted animals hyperpyrexic? 4. Strain variations in the leptin/brown adipose tissue pathway 5. Is obesity due to lack of obesity-induced thermogenesis? 6. Are age-induced obesity and cold sensitivity due to leptin resistance? 7. Activation of brown adipose tissue may not be a general mechanism for all food component deficiencies 8. Lipids containing polyunsaturated fatty acids activate in their own right 9. Does hyperphagia-induced thermogenesis exist? 10. Cancer cachexia and brown adipose tissue C. Influence of Sex Hormones on Brown Adipose Tissue 1. Androgen-induced thermogenesis 2. Estrogen-induced thermogenesis 3. Gestational brown-fat atrophy is caused by fetal heat production 4. Lactational atrophy is caused by lactational heat production D. Central Regulation of Metaboloregulatory Thermogenesis 1. All metaboloregulatory control may come together in the VMN 2. Effects of acute meal signals may be directly on the VMN 3. Leptin activates brown adipose tissue via the activating melanocortin system 4. Glucocorticoid inhibits leptin-sensitive cells 5. Further mediation of the leptin signal from the melanocortin receptors involves corticotropin-releasing factor 6. Serotonin from dorsal raphe and the action of weight-reducing agents 7. Lateral hypothalamic nucleus inhibits 8. The paraventricular nuclei mediate the brown fat-inhibitory NPY-borne signal VII. UPTAKES AND CLEARANCES A. Lipid Clearance and Brown Adipose Tissue 1. Triglyceride clearance through lipoprotein lipase activity 2. Passive effect of increased lipoprotein lipase activation on fatty acid composition of triglycerides and phospholipids B. Is Brown Adipose Tissue an Important Organ for Glucose Clearance? 1. Norepinephrine and GLUT(1?)-mediated glucose uptake 2. Insulin and GLUT4-mediated glucose uptake C. During Nonshivering Thermogenesis, Brown Adipose Tissue Is the Major Oxygen-Consuming Organ in the Body VIII. BROWN ADIPOSE TISSUE AS A SECRETORY ORGAN A. Autocrine 1. Basement membrane proteins 2. Adipsin 3. Basic fibroblast growth factor 4. Insulin-like growth factor I 5. Prostaglandins 6. Adenosine B. Paracrine 1. Nerve growth factor 2. VEGF 3. Nitric oxide and blood flow 4. Angiotensinogen C. Endocrine 1. Fatty acids 2. Leptin, adiponectin, and resistin 3. T3 4. Is an antiobesity factor secreted from brown adipose tissue? 5. Heat IX. SIGNIFICANCE OF BROWN ADIPOSE TISSUE FOR HUMANS AND OTHER MAMMALS A. Brown Adipose Tissue and Humans 1. Norepinephrine-induced thermogenesis in humans 2. A cure for obesity? B. Benefits of Nonshivering Thermogenesis
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ABSTRACT |
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I. A MAMMALIAN PREROGATIVE: BROWN ADIPOSE TISSUE |
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In contrast to other mammalian organs, brown adipose tissue is still scientifically a rather new organ. Although described in certain mammals since 1551 (244), the realization that brown adipose tissue is found in all mammals has occurred within the last century. That heat production is one of the functions of brown adipose tissue has only been formulated for 40 years (751), and the involvement of the tissue in or even its full responsibility for diverse types of metabolic inefficiency (i.e., as a possible antiobesity organ) has only been discussed for some 20 years (680). The identification of UCP1 as the mitochondrial protein responsible for the unique function of brown adipose tissue is of a similar short age (19, 311).
The present review is a further contribution to a series of reviews and books on nonshivering thermogenesis and brown adipose tissue (332, 379, 446, 575, 754, 816). Very detailed reviews of brown adipose tissue function in general, especially in connection with metabolic control, were compiled in the late 1980s (328, 329, 331), and we will not here replicate these efforts.
Brown adipose tissue morphology has been particularly elegantly presented recently (133), but the impacts of the scientific developments of the last decade have not been synthesized into a comprehensive analysis of brown adipose tissue function. The last decade has brought us an understanding of the background of genetically obese phenotypes, the identification of a family of mitochondrial carrier proteins (659) more similar to UCP1 than to any other protein (raising questions concerning the uniqueness of brown fat-derived thermogenesis and metabolic inefficiency) and, as new experimental tools, the development of mice strains deficient in brown adipose tissue (462) or in UCP1 (200), which in their turn have allowed for the demonstration of the essentiality of UCP1 for thermogenesis in the brown adipocytes (491) and for nonshivering thermogenesis in the intact animal (260, 262, 565). We have thus in the present review concentrated on issues developing during the last decade. With now more than 5,000 articles dealing to some extent with brown adipose tissue as such, there is no possibility to be comprehensive, nor is it possible to encompass general biological concepts; we attempt only to reference observations made specifically in brown adipose tissue. We have nevertheless attempted to be conclusive, in the light of available evidence, often to the exclusion of occasional contradictory evidence (which, perhaps, may ultimately become the more correct interpretation); we consequently apologize for misinterpretations, oversights, and omissions.
Certain types of experiments on brown adipose tissue are often performed on particular species of animals, e.g., mice, rats, and Syrian and Djungarian hamsters. We have tried to avoid qualifying each statement as to species, strain, or other condition investigated, provided we have not considered this type of qualification essential. We thus discuss a generic brown adipose tissue. We concentrate especially on the functional significance of brown fat-derived thermogenesis, i.e., to what extent are alterations in metabolism and metabolic efficiency, observed under a broad variety of physiological conditions, explainable through brown adipose tissue thermogenic activity (see sects. V and VI). However, to be able to do this, we initially describe the thermogenic mechanism in the single heat-producing unit, the brown adipocyte, and how the functional capacity of brown adipose tissue may be altered (i.e., the recruitment processes) (see sects. II–IV). We also discuss the extent to which brown adipose tissue may play a systemically important role in other respects than thermogenesis, by releasing or extracting substances to or from the circulation (see sects. VII and VIII), and we finalize with a short comment (see sect. IX) on brown adipose tissue function in the mammalian species that attracts much of our interest: humans.
To facilitate the more detailed discussion that will follow, a general overview of brown adipose tissue function within the mammalian organism can be seen in Figure 1A. Although the thermogenic unit is the brown adipocyte, placed in the center of Figure 1, it is evident from the figure that even within the tissue, the brown adipocyte cannot work in isolation: its activity is controlled by the nerve fibers reaching each cell, and the brown adipocyte is dependent on adequate delivery of oxygen and substrate (lipids) through the capillaries surrounding each cell (212); the delivery of its product, heat, to the organism is equally dependent on the heated blood leaving the tissue. Thus, although the brown adipocytes themselves constitute the main volume of the tissue, the mature brown adipocytes are probably in minority among the cells in the tissue (241), with the largest number of cells being the endothelial cells of the capillaries, and the interstitial cells and preadipocytes that, under conditions of increased thermogenic demand, will divide and differentiate to form new brown adipocytes. In such recruitment phases, not only the number of brown adipocytes but also the capillaries and the nerve terminals have to expand in a coordinated way to fulfil the new demands.
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The study of the physiological signficance of the tissue would have been much simpler if brown adipose tissue was only found in one place in the body. However, as summarized in Figure 1B, brown adipose tissue is found in defined but dispersed areas in the body, and brown adipocytes may be identified in clusters even within white adipose tissue depots, to a varying degree in different animals or strains of animals. Therefore, the metabolic significance of the tissue in different physiological conditions is still not fully established, but as will be evidenced in the present review, it is an organ with unique functions.
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II. NOREPINEPHRINE CONTROLS THE THERMOGENIC PROCESS |
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Among the factors that influence the brown adipocyte, norepinephrine is both the most important and the most well-studied. This effector is most significant physiologically, not only for the acute thermogenic process but also for the control of cell proliferation, advanced cell differentiation, and apoptosis. We therefore first review adrenergic signaling in brown adipocytes, leading towards regulation of the acute thermogenic process. Adrenergic effects on cell proliferation, differentiation, and apoptosis are discussed in section III.
A. Norepinephrine Signaling Through 
3-Receptors Leads to Thermogenesis
1. 3-Adrenoceptors in mature brown adipocytes
In mature brown adipocytes, norepinephrine interacts with all three types of adrenergic receptors: , 
2, and 1; these receptor types are associated with activation of different signaling pathways in the brown adipocytes, as will be detailed below. The most significant and the most studied pathway is the pathway for -adrenergic stimulation of thermogenesis (Fig. 2).
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Of the three subtypes of -adrenergic receptors, the 3-adrenoceptor is the most significant in mature brown adipocytes from rodents. 1-Adrenoceptors are also expressed in mature brown adipocytes, but they are not coupled to any significant extent to signaling processes in these cells; they are, however, coupled to cAMP production in brown preadipocytes (76) (see sect. IIIA), which means that in membrane preparations from total brown adipose tissue, both receptor subtypes will be functional (126). 2-Adrenoceptors are not expressed in the brown adipocytes themselves (46), but they are expressed in the tissue (651, 652) and can be observed as binding sites in membrane preparations from brown adipose tissue (438, 676). These 2-adrenoceptors are probably predominantly localized to the vascular system.
The extent to which the 3-adrenoceptor mediates the physiological effects of norepinephrine is routinely examined by comparing the effects of norepinephrine stimulation with those of a "specific" 3-agonist. The 3-agonists most commonly used are BRL-37344 (25) (which, however, is only a selective 3-agonist, i.e., at higher concentrations it also stimulates 2-receptors), CGP-12177 (516) (which is an antagonist on 1/2-receptors), and CL-316243 (336) (which must be considered presently as the most selective 3-agonist available). It is generally assumed that thermogenesis stimulated by one of these agents (especially CL-316243) in intact animals is indicative of brown adipose tissue thermogenesis, primarily because 3-receptors are practically only found in white and brown adipose tissue, and because the total thermogenic capacity of white adipose tissue is supposedly so low that it can be neglected in this context [but this assumption has been challenged (275a)].
The existence of a fourth -adrenoreceptor, the 4-receptor, has sometimes been discussed (234, 391), also in brown adipose tissue (626). One of the properties of this receptor should be that it is stimulated by CGP-12177 (it is thus difficult to differentiate from the 3-receptor in normal brown adipocytes). Such a "4-effect" has sometimes been ascribed to an atypical activation by CGP-12177 of 1-receptors in a certain conformation (rather unexpectedly, as CGP-12177 is a high-affinity antagonist on these receptors) (275, 408). There is thus no gene for this "4-receptor," and the phenomenon is still not fully clarified.
In addition to being characterized by specific stimulation by "specific" 3-agonists, 3-adrenoceptors are also characterized by a very low affinity for classical -adrenergic antagonists, such as propranolol [with a pA2 of 
9 on 1/2-receptors and 6 on 3-receptors, i.e., about 3 orders of magnitude lower affinity (25, 900)]. To eliminate 3-stimulation in vivo, very high concentrations of propranolol must therefore be used (at least 
10 mg/kg body wt). Unfortunately, no well-recognized high-affinity selective 3-antagonist is presently available; SR 59230A has been suggested (585), but the efficacy of this ligand has been criticized. Thus simple questions concerning the significance of the 3-pathway cannot be answered simply, by experimentally inhibiting this pathway selectively.
2. 3-Adrenoceptors do not possess properties essential for brown adipose tissue function
The distinct localization of 3-receptors to brown and white adipose tissue has led to suggestions that the receptors as such may have functional properties necessary or at least advantageous for (brown) adipose tissue function. This does not, however, seem to be the case.
In this respect, it is noteworthy that the guinea pig lacks identifiable, functional 3-receptors in brown adipose tissue (33), but its brown adipose tissue is nonetheless fully functional (80, 338, 456). Similarly, brown adipocytes prepared from animals in which the 3-gene has been ablated are fully functional, except that in these cells, it is the 1-adrenoceptor that mediates the -response (139, 408, 596). The ability of brown adipocytes from 3-ablated animals to respond to norepinephrine via 1-receptors does not indicate that 1-receptors are normally responsible for stimulation of thermogenesis; rather, there is an induced expression of 1-adrenoceptors in these animals (781). [The term compensatory is often used for such a situation, but this is easily interpreted as implying some nearly conscious act on the part of the cell; however, the increase in 1-adrenoceptor expression is probably coincidental, as expression of the 1-gene is under positive adrenergic control (46) and is thus self-inducing under conditions of increased sympathetic tone, which would be expected to occur when insufficient heat is produced due to the absence of 3-receptors.]
The 3-receptors distinguish themselves from the 1/2-receptors by lacking most of the amino acid residues that are normally thought to be involved in receptor desensitization (199, 545). It can easily be argued that it would be advantageous for brown adipocytes to possess receptors that were not easily desensitized (because thermogenesis often has to proceed for very prolonged periods). It could therefore be assumed that cells with 1-receptors would desensitize more rapidly than 3-expressing wild-type cells, although there is no published evidence for this. In this context, it is also notable that, although the 3-receptor may not be easily experimentally desensitized, the 3-expression level (mRNA) is dramatically downregulated (at least transiently) during continuous adrenergic stimulation (47, 276, 398), and this could also result in functional desensitization.
It is sometimes stated that 3-receptors are less sensitive to norepinephrine than are 1-receptors. Thus it has been claimed that at low levels of sympathetic stimulation, it would be the 1-adrenoceptors that would be activated (32, 233, 421). There is, however, no unequivocal evidence for this, neither in transfected systems (in which 3-receptors have affinities intermediate between 1- and 2-receptors, Ref. 797), nor functionally in brown adipocytes [the functional EC50 for cAMP formation by norepinephrine in preadipocytes (where the 1-receptor is dominant) is not lower than it is in mature brown adipocytes (76)]. [There is, however, a lower sensitivity of the 3-receptor than the 1-receptor for the pharmacological -agonist isoprenaline (isoproterenol) (596).] At low norepinephrine concentrations, the thermogenic response is more sensitive to a given dose of propranolol than it is at high norepinephrine concentrations (32), but this is an inherent feature of interaction between agonists and antagonists and does not indicate a shift from 1-receptors at low to 3-receptors at high norepinephrine.
It has also been suggested that 3-receptors could have a dual coupling to the transducing G proteins (to Gi as well as to Gs, see below), but as the splice variant expressed in mouse brown adipose tissue does not have this property (363a), this would seem not to be a general phenomenon in native brown adipocytes.
Thus, at present, there is no evidence that the presence of 3-receptors (as compared with 1/2-receptors) on brown adipocytes and their coupling to thermogenesis is anything other than coincidental, and the 3-receptor apparently does not confer to the brown adipocytes any demonstrated physiological advantage. However, the presence of the 3-receptors predominantly (although not exclusively) on white and brown adipocytes means that these receptors are potentially convenient targets for drugs against obesity, even bearing in mind the lower functional significance of these receptors in human than rodent adipose tissue.
3. Only Gs proteins couple to thermogenesis
-Adrenergic receptors normally couple to G proteins of the Gs subtype. This coupling has been indirectly demonstrated in brown adipose tissue, since norepinephrine infusion enhances the ability of cholera toxin to ADP-ribosylate the Gs protein (274) and cholera toxin can mimic the effects of -stimulation (479). Gs proteins exist in GsL and GsS forms in brown adipocytes as in other tissues; during differentiation from brown preadipocytes to mature brown adipocytes, the GsS variant increases, without any change in GsL (72) and without any functional change being observed.
Based mainly on experiments with ectopically expressed human 3-receptors and in cell lines, a particular feature of 3-adrenoceptors has been suggested to be that they may be dually coupled, i.e., not only to Gs but also to the inhibitory Gi proteins (127, 243, 757). This has been discussed also as being a component in the further mediation of the 3-signal to the mitogen-activated protein (MAP) kinase system(s) (see below). A parallel 3-stimulation of Gi would mean that the signal (in the form of cAMP formation) would be self-limiting, and the inhibitor of the Gi pathway, pertussis toxin, should in this case specifically increase 3-induced cAMP formation in brown adipocytes. However, whereas pertussis toxin does increase cAMP formation, it does so independently of which receptor, adrenergic or not, that cAMP formation is stimulated through (unpublished observations). Thus a high inherent Gi stimulation (endogenous or due to an unknown agonist) may constitutively inhibit cAMP formation. Indeed, when pertussis toxin is given in vivo, a large left-shift of norepinephrine sensitivity is observed (785).
4. Adenylyl cyclase, cAMP, and protein kinase A mediate the thermogenic signal
The further -adrenergic signaling cascade is mediated via adenylyl cyclase activation: the norepinephrine-induced cAMP formation is fully mediated via 3-receptors in mature brown adipocytes (899, 900). Correspondingly, all tested -adrenergic effects, including thermogenesis (144, 709, 778), can be mimicked by the adenylyl cyclase activator forskolin. It is not fully established which of the 10 adenylyl cyclase isoforms that are responsible for mediating the signal in mature brown adipocytes; several are expressed in brown adipose tissue (125, 128), and there are functional indications of a change in active adenylyl cyclase isoform during brown adipocyte differentiation (78).
In other tissues, in addition to its interaction with protein kinase A, cAMP directly activates other proteins [cation channels, exchange proteins directly activated by cAMP (EPACs)]. There is no indication to date that any cAMP effects in brown adipocytes are mediated in ways other than through activation of protein kinase A, the activity of which is increased as an effect of adrenergic stimulation (801); conversely, the inhibitor of protein kinase A, H-89, blocks all effects of 3-stimulation so far identified and examined in native brown adipocytes (thermogenesis, downstream kinases, gene expression) (107, 226, 227, 450).
5. Protein kinase A-phosphorylated proteins
Through phosphorylation of a series of target enzymes, the activated protein kinase A leads to further mediation of the adrenergic signal.
A) PHOSPHORYLATION OF NUCLEAR-RELATED PROTEINS. Also in brown adipocytes, protein kinase A phosphorylates the transcription factor CREB (802). CREB then supposedly activates the expression of genes, including that for UCP1 (see sect. IIIB3) (Fig. 2). Phosphorylated CREB also induces expression of the transcription factor ICER (801), which is competitive with CREB itself on (certain) CRE sites where it instead acts as a repressor. This successive increase in ICER formation may explain the transient expression of certain genes occurring during sustained norepinephrine stimulation.
The protein kinase A pathway also leads to activation of Src (450), but this cannot be direct, as Src is phosphorylated on a tyrosine residue and is thus not a direct target of protein kinase A; activation of an intermediate tyrosine kinase must therefore be postulated. Activation of Src leads to subsequent activation of one of the three MAP kinase pathways, the Erk1/2 pathway (451, 739), which in its turn couples further to inhibition of apoptosis (451) (see sect. IIIC) (but, in contrast to the CREB pathway, is not linked to control of, e.g., UCP1 gene expression; Ref. 107).
Protein kinase A also induces the activation of a second MAP kinase pathway, the p38 pathway (107). This activation has been suggested to be involved in the adrenergic stimulation of UCP1 gene expression. The third MAP kinase pathway, the stress-activated JNK pathway, is not stimulated by norepinephrine in brown adipocytes in culture; activation is seen in the tissue in vivo during cold exposure, but the cell type and pathway for this activation are unknown (unpublished observations).
The coupling from G protein-coupled receptors (such as the 3-receptors) to MAP kinases has been proposed in other systems to proceed via a transactivation of a plasma membrane tyrosine kinase receptor, most often the epidermal growth factor (EGF) receptor [or the platelet-derived growth factor (PDGF) receptor] (161, 322, 492). Although the EGF receptor exists and is functional in brown adipocytes (318, 449), it is not activated (phosphorylated) following -adrenergic stimulation, and inhibition of its activity (by the EGF receptor inhibitor and ATP analog AG1478) does not inhibit norepinephrine-induced MAP kinase activation (although it inhibits EGF-induced MAP kinase activation) (449). There is therefore no indication that transactivation of the EGF receptor is an obligatory step in norepinephrine-induced MAP kinase activation.
B) PHOSPHORYLATION OF CYTOSOLIC PROTEINS. In parallel with its activation of the nuclear proteins summarized above, protein kinase A also phosphorylates (activates) a series of proteins in the cytosol. It probably activates the protein phosphatase inhibitor DARPP (502), in this way potentially prolonging its own action.
Protein kinase A probably also activates/inhibits a series of metabolic pathways in the brown adipocyte, but with the exception of the lipolytic pathway, such pathways have not been studied in brown adipocytes. However, the lipolytic pathway is the one that leads to thermogenesis in the brown adipocytes, and this pathway is therefore central to the understanding of control of thermogenesis in brown adipocytes (and thus of nonshivering thermogenesis in general).
B. Thermogenesis Is Due to Activation of UCP1 Through Lipolysis
Since the formulation of brown adipose tissue as a thermogenic organ (751), a number of molecular mechanisms to accomplish this thermogenesis have been proposed. These have included futile cycles in a broad sense, such as a lipolysis/esterification cycle or activation of Na+-K+-ATPase. These types of suggested mechanisms may be classified together as ATP dependent, since they require that ATP is formed and then used in an "unproductive" way (principally as in muscular shivering thermogenesis), leading to ADP generation and consequently to stimulation of substrate oxidation/oxygen consumption in the mitochondria (i.e., thermogenesis). However, an early observation that inhibition of ATP synthase (with oligomycin) only partly reduced norepinephrine-induced thermogenesis (629), as well as the successive realization that brown fat mitochondria generally have a remarkably low ATP synthase capacity (447) [due to a severe lack of the synthase complex (106, 356), which in its turn results from a specific lack of expression of one of the genes for subunit c, the P1 gene (14, 355)] has led to the conclusion that ATP-consuming mechanisms cannot be responsible for the thermogenic process in brown adipocytes.
The alternative formulation, that no ATP is formed and that oxidation is "uncoupled" (447, 755) in the sense that the word was originally used as describing "an oxidative process not coupled to ATP synthesis," has manifested itself in the identification of "the" uncoupling protein UCP1 (thermogenin). However, the identification of other proteins also classified, at least phylogenetically, as uncoupling proteins, such as UCP2 and UCP3 (see sect. IIB3C), has meant that even after the identification of UCP1, the question remained as to whether UCP1 is essential for all norepinephrine-induced thermogenesis in the brown adipocytes, or whether other mechanisms could contribute. In this respect, experiments with brown adipocytes isolated from UCP1-ablated mice (Fig. 3A) have been conclusive; they clearly demonstrate that in the absence of UCP1, no thermogenesis can be induced in brown adipocytes by norepinephrine (491). There is thus no reason to believe that any processes other than that mediated by UCP1 are by themselves thermogenic in brown adipocytes.
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1. Stimulation of lipolysis stimulates thermogenesis
It is a classical observation that the thermogenic process in brown adipocytes can be mimicked by the addition of fatty acids (630, 647) (Fig. 3B). That this fatty acid-induced thermogenesis is also completely UCP1 dependent (491) (Fig. 3B) makes it likely that, even physiologically, the activation of lipolysis is a sufficient trigger for initiation of thermogenesis in brown adipocytes. Indeed, all manipulations that induce lipolysis in brown adipocytes also induce thermogenesis, and no thermogenesis can be evoked without simultaneously evoking lipolysis.
Lipolysis, observed as glycerol or fatty acid release, is norepinephrine-induced in brown adipocytes (51, 206, 419, 568), just as is thermogenesis (208, 562, 630). Lipolysis is stimulated through 3-receptors (25, 124) as is thermogenesis (900). Both processes occur downstream of cAMP formation, as they can be induced also by the adenylyl cyclase activator forskolin (207) or by the addition of cAMP analogs (144, 648, 827).
That lipolysis is due to protein kinase A activation can presently only be deduced indirectly, because thermogenesis is inhibited by the protein kinase A inhibitor H-89 (226). The stimulation of lipolysis is composed of two processes: activation of hormone-sensitive lipase (HSL) and phosphorylation (deactivation) of perilipin (Fig. 4).
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Brown adipocytes contain HSL (345), and it has normally been formulated that it is through the norepinephrine-induced phosphorylation of this enzyme that lipolysis is activated (although such phosphorylation has not been directly demonstrated in brown adipose tissue). However, the effect of adrenergic stimulation on lipolytic capacity, measured enzymatically as lipolysis of a triglyceride emulsion in vitro, is very marginal: only an 50% increase in lipolytic activity is induced by norepinephrine (733), a far lower degree of activation than would be expected.
This low degree of activation is probably explainable as an experimental limitation in this type of experiments. Artificial triglyceride emulsions are not endowed with perilipin, the protein that normally covers the triglyceride droplets within the cell (56). Perilipin protects the triglycerides against HSL activity (485). Activated protein kinase A phosphorylates perilipin (124), the phosphorylated perilipin is dissociated from the triglyceride droplets, and the droplets now become freely exposed to attack by HSL, which is translocated upon phosphorylation to the lipid droplets, at least in white adipose tissue (135, 528) (not as yet demonstrated in brown adipose tissue). This combination of lipase activation and perilipin inactivation may explain the large increase in lipolysis observed within the cell. In accordance with this, (white) adipocytes from perilipin-deficient mice display a high basal lipolysis that cannot be further activated by adrenergic stimulation, and in perilipin-deficient animals, brown adipose tissue appears very lipid depleted. Perilipin-deficient mice also display an increased basal metabolic rate. It is possible that the constitutively increased lipolysis in their brown adipocytes is sufficient to constitutively activate thermogenesis and that it is this extra thermogenesis that explains the lean phenotype of these mice (485) (although this has not been directly demonstrated).
That HSL is involved can also be seen from studies of HSL-deficient mice (608, 854). In these mice, basal lipolytic activity in brown adipose tissue is not diminished. However, catecholamine-induced lipolysis in white adipocytes is eliminated (854), implying a similar result in brown adipocytes. In these HSL-deficient animals, the white and brown adipocytes become heterogeneously more fat-filled than in wild type, further indicating that lipolysis is diminished (608, 854). The HSL-deficient mice are not more sensitive to an acute cold stress than are wild-type mice (608, 854), but this is not in itself evidence that brown adipose tissue is thermogenically active in these mice, despite the absence of HSL. (As discussed in section VB, acute defense against cold is mainly through shivering, and direct examination of brown adipose tissue thermogenic capacity would be required to conclude on a noninvolvement of HSL in the thermogenic response.) The most reasonable conclusion is thus still that HSL is both responsible and obligatory for norepinephrine-mediated lipolysis in brown adipocytes.
2. Fatty acids are the thermogenic substrates
Lipolysis of triglyceride droplets ultimately results in the liberation of glycerol and free fatty acids within the cell. Although some fatty acids may leave the cell (see sect. VIIIC1), most are channelled further within the cell. In the cytosol, they are probably bound to fatty acid-binding proteins. Similarly to other adipocytes, brown adipocytes possess the adipocyte form of the fatty acid binding protein, A-FABP or FABP4 (= aP2) (156). However, in contrast to white adipocytes, brown adipocytes also possess the heart form of this protein, H-FABP (156), and in contrast to what is the case for the A-FABP, gene expression of H-FABP is dramatically induced by norepinephrine. Although mRNA levels are not direct indicators of protein levels, it would seem likely that brown adipocytes possess very high levels of fatty acid binding proteins; accordingly, the level of free fatty acids in the cytosol is probably low, despite high rates of lipolysis.
Although some fatty acids may be degraded initially in peroxisomes (287, 556), most are channelled towards the mitochondria. In the mitochondrial environment, they may have several roles. They are definitely the substrate for thermogenesis, and they are most likely also involved in the regulation and/or function of UCP1.
In their fate as substrates, the fatty acids are transferred into the mitochondria via the general activation/carnitine shuttle system and are then -oxidized in the mitochondria, with the released acetyl CoA moities being oxidized in the citric acid cycle (Fig. 4). The catabolic pathway is thus not different from that in other cells (although brown adipocytes contain very high amounts of the catabolic enzymes involved, Ref. 218). Also similarly to what happens in the mitochondria of other cell types, the passage of the released electrons through the respiratory chain results in the pumping out from the mitochondrial matrix of protons and the establishment of a mitochondrial membrane potential (Fig. 4). Thus, in the catabolic steps, the mitochondria of brown adipose tissue are as energy-conserving as any other mitochondria. The difference, i.e., the thermogenic ability, results from the existence of high amounts of UCP1 in the mitochondria of brown adipose tissue.
3. The uncoupling protein UCP1
UCP1 (earlier known simply as UCP, or as the uncoupling protein, as thermogenin, as the GDP-binding protein, or as the 32,000-Da polypeptide) is a member of the mitochondrial carrier protein family. Present knowledge on UCP1 has been extensively reviewed (237, 397, 400, 560, 565, 569, 653). We will therefore here only summarize some of the more important points.
As a member of the mitochondrial carrier protein family, UCP1 shares many points of homology with the other members of this large family, including its tripartite structure and amino acid sequences, which are both conserved between the three 100-amino acid sequences and between many members of the mitochondrial carrier family; these features will not be discussed further here (65, 237, 565) (Fig. 5). There are also sequences and residues that are of particular interest for UCP1 function. One topological area is the amino acid residues involved in the binding of purine nucleotides (GDP in Fig. 5); these residues are also found in the sister proteins UCP2 and UCP3 (see sect. IIB4C). Other sequences of particular interest are two sequences that are fully conserved within UCP1 from all species as yet characterized but which are not found in any other mitochondrial carrier. These sequences are in the middle of the central loop (probably facing the matrix) and the last part of the COOH terminus (facing the cytosol). The COOH-terminal sequence is also immunogenic, and selective antibodies are preferably designed to react to this sequence. The actual function of these two conserved sequences is not known presently (although there are some indications concerning the histidines in the central loop, Refs. 188, 830a), but their unique and consistent presence in UCP1 indicates that they could be of importance for the functioning of this protein.
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A) HOW IS UCP1 ACTIVATED? The early observation that isolated brown fat mitochondria had high rates of respiration when examined under conditions in which "normal" mitochondria had low respiratory rates (i.e., in the presence of oxidizable substrate but the absence of ADP) indicated that an "uncoupling" mechanism existed in brown fat mitochondria. In a Mitchellian formulation, uncoupling in this sense must correspond to an increased proton permeability. The observation that GDP (or with similar, or even higher affinity, GTP, ADP, and ATP) could inhibit this high proton permeability (574) led experimentally to the identification of UCP1 (311) and also indicated that a physiological regulatory mechanism for UCP1 action must exist. It is generally accepted that brown fat mitochondria, and thus UCP1, are exposed in the resting state to cytosolic nucleotides and therefore not active (although some authors instead claim that the cytosolic nucleotides do not have this function and that UCP1 is inactive due to the absence of a necessary cofactor: free fatty acids). However, despite nearly four decades of experimentation on brown adipose tissue mitochondria and more than two decades of examination of the responsible protein UCP1, a full and generally accepted understanding of the control of proton (or proton equivalent) transport and the mechanism for this transport (which in some formulations may be said to be the same thing) has not been reached.
The tenet that fatty acids, either as such or as derivatives, are involved in the physiological activation of UCP1 and/or the transport mechanism, is generally accepted. The question is still what exactly the fatty acids do; there are presently at least three formulations: that they act as allosteric regulators, as cofactors, or as proton shuttles (Fig. 6).
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In the allosteric interaction model (Fig. 6A), the fatty acids interact with a site on UCP1 leading to its activation. In bioenergetic terms, the activation may be formulated as "lowering the threshold for a Zener diode" (653, 655), but what this means in molecular terms has not been clarified. The presumed fatty acid binding site has not been identified. The allosteric interaction model would become even simpler (competitive, orthosteric) if the free fatty acids could compete away the inhibitory purine nucleotides presumably bound to UCP1 in the resting state and in this way activate UCP1. There are, however, no indications that free fatty acids can do this. The fatty acid derivative acyl CoA ("activated" fatty acids) has the ability to compete with bound purine nucleotides and has been suggested as a competitive activator of UCP1 (105, 768, 769), but convincing evidence for a physiological relevance for such an effect is lacking.
In the cofactor theory (Fig. 6B), the fatty acids become localized to binding sites within the proton-conducting "channel" of UCP1, and their acid moieties then function as "stepping stones" for protons as they pass through the membrane (868); again, no interaction with the purine nucleotide-binding inhibitory site is formulated. The intrachannel fatty acid binding sites required for this model have not been identified.
In the shuttling theory (Fig. 6C), an observation by Skulachev (17) that other mitochondrial carriers (notably the ATP/ADP carrier), in the presence of free fatty acids, could function as uncoupling proteins has been extended by Garlid and Jezek (237), as being the mechanism also for UCP1 function. In this formulation, it is not protons that are transported by UCP1 over the mitochondrial membrane; rather, protons (re)enter the mitochondria in the form of the undissociated fatty acid, and the fatty acid, in its anionic form, (re)exits the mitochondria carried by UCP1. There is ample evidence that this process can occur in an experimental system (237, 373). The theory has been questioned (268), and objections can be raised of a more theoretical nature. The process as such is not specific for UCP1; rather, a series of mitochondrial carriers (not only the ATP/ADP carrier), physiologically expected to perform other tasks, can be convinced to function as "uncoupling proteins" under similar experimental conditions. If this is the case, it may be asked what the evolutionary advantage is of UCP1. Correspondingly, the conserved unique amino acid sequences (Fig. 5) imply acquisition of specific properties. Also, the role of the inhibitory GDP-binding site on UCP1 is presently unsolved in this model. Considering the high levels of fatty acid binding proteins in the cytosol of brown adipocytes, it may also be questioned as to whether fatty acids can reach sufficiently high free levels necessary for this process. Thus none of the three models presently proposed has been unequivocally demonstrated to fully explain the regulation and protonophoric properties of UCP1.
It was anticipated that examination of brown adipose tissue mitochondria from UCP1-ablated mice would solve some of these issues. Initial studies unexpectedly indicated that the difference in fatty acid sensitivity between brown fat mitochondria with or without UCP1 was minor (341, 490, 566). However, with more optimal substrate (i.e., pyruvate or fatty acids in the oxidizable form of palmitoyl carnitine) and by expressing the effect as a function of the free fatty acid level (rather than the added), quite marked effects of the presence of UCP1 are noticable, with UCP1-containing mitochondria being more than 10-fold more fatty acid sensitive than mitochondria without UCP1 (721a). Remarkably, this UCP1-dependent thermogenesis induced by free fatty acids in brown fat mitochondria is competitive with GDP. Because no direct competition between fatty acids and the GDP-binding site exists, the competition must be functional. A formulation by Rial and Nicholls (654) suggests that UCP1 is transformed by free fatty acids and by GDP into two different states (Fig. 6D), and this could result in the apparent competition observed.
B) UCP1 IN TISSUES OTHER THAN BROWN ADIPOSE TISSUE. It has long been the general notion that UCP1 expression is a unique feature of brown adipose tissue, indeed to the degree that "brown adipose tissue" may be defined as an (adipose) tissue that has the ability to express UCP1; the word ability indicates that actual expression is not necessarily seen, e.g., in brown preadipocytes and even in nonstimulated otherwise differentiated brown adipocytes. There have, however, been occasional reports that UCP1 is found in non-brown adipose tissues.
Concerning reports that UCP1 is expressed in "white" adipose tissue depots (140, 238, 288, 411, 593, 822, 882), this seems to be mainly a question of definition. We prefer to formulate it that any adipocyte that has the ability to express UCP1 is a brown adipocyte, and the occurrence of UCP1 in white adipose tissue therefore does not constitute any change in paradigm, but merely indicates that brown adipocytes can occur sporadically in predominantly white depots.
The situation is more complex in nonadipose tissues. Early reports that UCP1 mRNA was observable in liver of newborn and cold-exposed rats (741) resulted from un-specificity of the cDNA clone used (666). Two reports indicating that chronic stimulation of animals with 3-agonists leads to UCP1 expression in skeletal muscle (541, 884) were published about the time when it was becoming clear that proteins closely related to UCP1 (i.e., UCP2 and UCP3, see below) were expressed in skeletal muscle; the observations have not as yet been confirmed under experimental circumstances ensuring that cross-reactivity of these UCP1-like proteins could not be the cause of the positive reactions seen. Indeed, chronic treatment of muscle cells (the cell line L6) with 3-agonists leads to increased expression of UCP2 (542). Thus it seems presently unlikely that UCP1 can be expressed in skeletal muscle following physiological or pharmacological stimulation.
Evidence, so far unconfirmed, has also been presented that UCP1 is expressed in a few, specific cells within the longitudinal muscle layer of (all) peristaltic organs in the body (573); this thus includes the entire gastrointestinal tract, urethra, as well as gonadal tissues (epidydimis and vas deferens in males and uterus in females). The evidence has been criticized (692a) and the reported total expression level in these tissues would in any case be very low at the protein level: in isolated mitochondria, only 1/1,000 of the level in brown-fat mitochondria and thus probably even a further factor of 10 lower on an organ basis, due to the low density of mitochondria compared with brown adipose tissue. It is therefore unlikely that this possible extra-adiposal UCP1 expression has any measureable significance for thermogenesis on a whole body basis. Indeed, classical observations demonstrate that animals that have been functionally eviscerated (i.e., the blood flow to all peritoneal organs cut off) respond to cold exposure or norepinephrine injection with a thermogenic response quantitatively identical to that of intact animals (170, 171). This demonstrates that even if UCP1 is present in peristaltic organs it is not of thermogenic significance. In the following we therefore make the assumption that UCP1-dependent thermogenesis of systemic significance entirely emanates from UCP1 in brown adipose tissue.
Whether this possible extra-adipose expression of UCP1 has any functional significance is not known. Adrenergically induced relaxation of precontracted intestinal strips is somewhat impaired in UCP1-ablated mice (722), but whether this is a secondary effect to the absence of UCP1 in brown adipose tissue or a demonstration of a genuine intestinal UCP1 effect is not settled as yet. However, interpretation of results on the expression of transgenes under the control of the UCP1 promoter (such as diphtheria to
