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The issue under discussion here is whether a decrease in the degree of UCP1 activity (and brown adipose tissue activity in general) could be a cause of obesity in humans. This possibility principally requires the existence of the phenomenon of diet-induced thermogenesis. Obesity could be a consequence of a reduced functionality of diet-induced thermogenesis. Experiments in mice indicate that diet-induced thermogenesis exists and is dependent on the presence of UCP1 and thus of brown adipose tissue activity. Accordingly, many (but not all) experiments indicate that in the absence of UCP1, mice become obese. Whether similar mechanisms exist in humans is still unknown. A series of studies have indicated a correlation between obesity and low brown adipose tissue activity, but it may be so that the obesity itself may influence the estimates of brown adipose tissue activity (generally glucose uptake), partly explaining the relationship. Estimates of brown adipose tissue catabolizing activity would seem to indicate that it may possess a capacity sufficient to help maintain body weight, and obesity would thus be aggravated in its absence. This article is part of a discussion meeting issue 'Causes of obesity: theories, conjectures and evidence (Part II)'.

Given the presence of brown adipose tissue in adult humans, an important issue is whether human brown adipose tissue is recruitable. Cold exposure is the canonical recruitment treatment; however, in experimental animals (mice), recruitment of brown adipose tissue is normally induced by placing the mice in constant cold, a procedure not feasible in humans. For possible translational applications, we have therefore investigated whether shorter daily excursions from thermoneutrality would suffice to qualitatively and quantitatively induce recruitment in mice. Mice, housed at thermoneutrality (30 °C) to mimic human conditions, were transferred every day for 4 weeks to cool conditions (18 °C), for 0, 15, 30, 120 and 420 min (or placed constantly in 18 °C). On the examination day, the mice were not exposed to cold. Very short daily exposures (≤30 min) were sufficient to induce structural changes in the form of higher protein density in brown adipose tissue, changes that may affect the identification of the tissue in e.g. computer tomography and other scan studies. To estimate thermogenic capacity, UCP1 protein levels were followed. No UCP1 protein was detectable in inguinal white adipose tissue. In the interscapular brown adipose tissue, a remarkable two-phase reaction was seen. Very short daily exposures (≤30 min) were sufficient to induce a significant increase in total UCP1 levels. For attainment of full cold acclimation, the mice had, however, to remain exposed to the cold. The studies indicate that marked alterations in brown adipose tissue composition can be induced in mammals through relatively modest stimulation events., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Concerning diet-induced thermogenesis, methodological issues relate mainly to the interpretation of measurements, rather than to the technical methodology as such. In the following, we point to a series of issues where the analysis often suggests the occurrence of UCP1-related diet-induced thermogenesis but where the observations are often the consequences of a process that has induced leanness rather than being the cause of them. We particularly emphasize the necessity of focusing on the total organism when interpreting biochemical and molecular data, where the concept of total tissue values rather than relative data better reflects physiologically important alterations. We stress the importance of performing experiments at thermoneutrality in order to obtain clinically relevant data and stress that true thermogenic agents may be overlooked if this is not done., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cold- and diet-induced recruitment of brown adipose tissue (BAT) and the browning of white adipose tissue (WAT) are dynamic processes, and the recruited state attained is a state of dynamic equilibrium, demanding continuous stimulation to be maintained. An involvement of macrophages, classical proinflammatory (M1) or alternatively activated anti-inflammatory (M2), is presently discussed as being an integral part of these processes. If these macrophages play a mediatory role in the recruitment process, such an involvement would have to be maintained in the recruited state. We have, therefore, investigated whether the recruited state of these tissues is associated with macrophage accretion or attrition. We found no correlation (positive or negative) between total UCP1 mRNA levels (as a measure of recruitment) and proinflammatory macrophages in any adipose depot. We found that in young chow-fed mice, cold-induced recruitment correlated with accretion of anti-inflammatory macrophages; however, such a correlation was not seen when cold-induced recruitment was studied in diet-induced obese mice. Furthermore, the anti-inflammatory macrophage accretion was mediated via β 1 /β 2 -adrenergic receptors; yet, in their absence, and thus in the absence of macrophage accretion, recruitment proceeded normally. We thus conclude that the classical recruited state in BAT and inguinal (brite/beige) WAT is not paralleled by macrophage accretion or attrition. Our results make mediatory roles for macrophages in the recruitment process less likely. NEW & NOTEWORTHY A regulatory or mediatory role-positive or negative-for macrophages in the recruitment of brown adipose tissue is presently discussed. As the recruited state in the tissue is a dynamic process, maintenance of the recruited state would need persistent alterations in macrophage complement. Contrary to this expectation, we demonstrate here an absence of alterations in macrophage complement in thermogenically recruited brown-or brite/beige-adipose tissues. Macrophage regulation of thermogenic capacity is thus less likely.

Animals that lack the hormone leptin become grossly obese, purportedly for 2 reasons: increased food intake and decreased energy expenditure (thermogenesis). This review examines the experimental evidence for the thermogenesis component. Analysis of the data available led us to conclude that the reports indicating hypometabolism in the leptin-deficient ob/ob mice (as well as in the leptin-receptor-deficient db/db mice and fa/fa rats) derive from a misleading calculation artefact resulting from expression of energy expenditure per gram of body weight and not per intact organism. Correspondingly, the body weight-reducing effects of leptin are not augmented by enhanced thermogenesis. Congruent with this, there is no evidence that the ob/ob mouse demonstrates atrophied brown adipose tissue or diminished levels of total UCP1 mRNA or protein when the ob mutation is studied on the inbred C57BL/6 mouse background, but a reduced sympathetic nerve activity is observed. On the outbred "Aston" mouse background, brown adipose tissue atrophy is seen, but whether this is of quantitative significance for the development of obesity has not been demonstrated. We conclude that leptin is not a thermogenic hormone. Rather, leptin has effects on body temperature regulation, by opposing torpor bouts and by shifting thermoregulatory thresholds. The central pathways behind these effects are largely unexplored., (© Endocrine Society 2019.)

Objectives: High-esterified pectin (HEP) is a prebiotic able to modulate gut microbiota, associated with health-promoting metabolic effects in glucose and lipid metabolism and adipostatic hormone sensitivity. Possible effects regulating adaptive thermogenesis and energy waste are poorly known. Therefore, we aimed to study how physiological supplementation with HEP is able to affect microbiota, energy metabolism and adaptive thermogenic capacity, and to contribute to the healthier phenotype promoted by HEP supplementation, as previously shown. We also attempted to decipher some of the mechanisms involved in the HEP effects, including in vitro experiments., Subjects and Experimental Design: We used a model of metabolic malprogramming consisting of the progeny of rats with mild calorie restriction during pregnancy, both under control diet and an obesogenic (high-sucrose) diet, supplemented with HEP, combined with in vitro experiments in primary cultured brown and white adipocytes treated with the postbiotic acetate., Results: Our main findings suggest that chronic HEP supplementation induces markers of brown and white adipose tissue thermogenic capacity, accompanied by a decrease in energy efficiency, and prevention of weight gain under an obesogenic diet. We also show that HEP promotes an increase in beneficial bacteria in the gut and peripheral levels of acetate. Moreover, in vitro acetate can improve adipokine production, and increase thermogenic capacity and browning in brown and white adipocytes, respectively, which could be part of the protection mechanism against excess weight gain observed in vivo., Conclusion: HEP and acetate stand out as prebiotic/postbiotic active compounds able to modulate both brown-adipocyte metabolism and browning and protect against obesity.

A reduction in the thermogenic activity of brown adipose tissue (BAT) is presently discussed as a possible determinant for the development of obesity in humans. One group of endogenous factors that could potentially affect BAT activity is the glucocorticoids (e.g. cortisol). We analyse here studies examining the effects of alterations in glucocorticoid signaling on BAT recruitment and thermogenic capacity. We find that irrespective of which manipulation of glucocorticoid signaling is examined, a seemingly homogeneous picture of lowered thermogenic capacity due to glucocorticoid stimulation is apparently obtained: e.g. lowered uncoupling protein 1 (UCP1) protein levels per mg protein, and an increased lipid accumulation in BAT. However, further analyses generally indicate that these effects result from a dilution effect rather than a true decrease in total capacity; the tissue may thus be said to be in a state of pseudo-atrophy. However, under conditions of very low physiological stimulation of BAT, glucocorticoids may truly inhibit Ucp1 gene expression and consequently lower total UCP1 protein levels, but the metabolic effects of this reduction are probably minor. It is thus unlikely that glucocorticoids affect organismal metabolism and induce the development of obesity through alterations of BAT activity., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: Hyperthyroidism is associated with increased metabolism ("thyroid thermogenesis") and elevated body temperature, often referred to as hyperthermia. Uncoupling protein-1 (UCP1) is the protein responsible for nonshivering thermogenesis in brown adipose tissue. We here examine whether UCP1 is essential for thyroid thermogenesis., Methods: We investigated the significance of UCP1 for thyroid thermogenesis by using UCP1-ablated (UCP1 KO) mice. To avoid confounding factors from cold-induced thermogenesis and to approach human conditions, the experiments were conducted at thermoneutrality, and to resemble conditions of endogenous release, thyroid hormone (thyroxine, T4) was injected peripherally., Results: Both short-term and chronic thyroxine treatment led to a marked increase in metabolism that was largely UCP1-independent. Chronic thyroxine treatment led to a 1-2 °C increase in body temperature. This increase was also UCP1-independent and was maintained even at lower ambient temperatures. Thus, it was pyrexia, i.e. a defended increase in body temperature, not hyperthermia. In wildtype mice, chronic thyroxine treatment induced a large relative increase in the total amounts of UCP1 in the brown adipose tissue (practically no UCP1 in brite/beige adipose tissue), corresponding to an enhanced thermogenic response to norepinephrine injection. The increased UCP1 amount had minimal effects on thyroxine-induced thermogenesis and pyrexia., Conclusions: These results establish that thyroid thermogenesis is a UCP1-independent process. The fact that the increased metabolism coincides with elevated body temperature and thus with accelerated kinetics accentuates the unsolved issue of the molecular background for thyroid thermogenesis., (Copyright © 2019 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The attractive tenet that recruitment and activation of brown adipose tissue (BAT) and uncoupling protein 1 (UCP1) could counteract the development of obesity and its comorbidities in humans has been experimentally corroborated mainly by experiments demonstrating that UCP1-ablated mice on a C57Bl/6 background (exempt from thermal stress) become more obese when fed a high-fat diet. However, concerns may be raised that this outcome of UCP1 ablation is restricted to this very special inbred and particularly obesity-prone mouse strain. Therefore, we have examined to which degree UCP1 ablation has similar metabolic effects in a mouse strain known to be obesity resistant: the 129S strain. For this, male 129S2/sv or 129SV/Pas mice and corresponding UCP1-knockout mice were fed chow or a high-fat or a cafeteria diet for 4 wk. The absence of UCP1 augmented obesity (weight gain, body fat mass, %body fat, fat depot size) in high-fat diet- and cafeteria-fed mice, with a similar or lower food intake, indicating that, when present, UCP1 indeed decreases metabolic efficiency. The increased obesity was due to a decrease in energy expenditure. The consumption of a high-fat or cafeteria diet increased total BAT UCP1 protein levels in wild-type mice, and correspondingly, high-fat diet and cafeteria diet-fed mice demonstrated increased norepinephrine-induced oxygen consumption. There was a positive correlation between body fat and total BAT UCP1 protein content. No evidence for diet-induced adrenergic thermogenesis was found in UCP1-ablated mice. Thus, the obesity-reducing effect of UCP1 is not restricted to a particular, and perhaps not representative, mouse strain.

The possibility that recruitment and activation of brown adipose tissue (BAT) thermogenesis could be beneficial for curtailing obesity development in humans prompts a need for a better understanding of the control of these processes [that are often referred to collectively as diet-induced thermogenesis (DIT)]. Dietary conditions are associated with large changes in blood-borne factors that could be responsible for BAT recruitment, but BAT is also innervated by the sympathetic nervous system. To examine the significance of the innervation for DIT recruitment, we surgically denervated the largest BAT depot, i.e., the interscapular BAT depot in mice and exposed the mice at thermoneutrality to a high-fat diet versus a chow diet. Denervation led to an alteration in feeding pattern but did not lead to enhanced obesity, but obesity was achieved with a lower food intake, as denervation increased metabolic efficiency. Conclusively, denervation totally abolished the diet-induced increase in total UCP1 protein levels observed in the intact mice, whereas basal UCP1 expression was not dependent on innervation. The denervation of interscapular BAT did not discernably hyper-recruit other BAT depots, and no UCP1 protein could be detected in the principally browning-competent inguinal white adipose tissue depot under any of the examined conditions. We conclude that intact innervation is essential for diet-induced thermogenesis and that circulating factors cannot by themselves initiate recruitment of brown adipose tissue under obesogenic conditions. Therefore, the processes that link food intake and energy storage to activation of the nervous system are those of significance for the further understanding of diet-induced thermogenesis.

In mice living under normal animal house conditions, the brown adipocytes in classical brown adipose tissue depots are already essentially fully differentiated: UCP1 mRNA and UCP1 protein levels are practically saturated. This means that any further recruitment - in response to cold exposure or any other browning agent - does not result in significant augmentation of these parameters. This may easily be construed to indicate that classical brown adipose tissue cannot be further recruited. However, this is far from the case: the capacity for further recruitment instead lies in the ability of the tissue to increase the number of brown-fat cells, a remarkable and highly controlled physiological recruitment process. We have compiled here the available data concerning the unique ability of norepinephrine to increase cell proliferation and inhibit apoptosis in brown adipocytes. Adrenergically stimulated cell proliferation is fully mediated via β 1 -adrenoceptors and occurs through activation of stem cells in the tissue; intracellular mediation of the signal involves cAMP and protein kinase A activation, but activation of Erk1/2 is not part of the pathway. Apoptosis inhibition in brown adipocytes is induced by both β- and α 1 -adrenergic receptors and here the intracellular pathway includes Erk1/2 activation. This unique ability of norepinephrine to increase cell number in an apparently mitogenically dormant tissue provides possibilities to augment the metabolic capacity of brown adipose tissue, also for therapeutic purposes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Much current understanding of brown adipocyte development comes from in-vitro cell models. Serum type may affect the behavior of cultured cells and thus conclusions drawn. Here, we investigate effects of serum type ("fetal bovine" versus "newborn calf") on responses to differentiation inducers (the PPARγ agonist rosiglitazone or the neurotransmitter norepinephrine) in cultured primary brown adipocytes. Lipid storage was enhanced by fetal versus newborn serum. However, molecular adipose conversion (Pparg2 and Fabp4 expression) was not affected by serum type. Rosiglitazone-induced (7-days) expression of thermogenic genes (i.e. Ucp1, Pgc1a, Dio2 and Elovl3) was not systematically affected by serum type. However, importantly, acute (2 h) norepinephrine-induced thermogenic gene expression was overall markedly higher (and adipose genes somewhat lower) in cells cultured in newborn serum. Thus, newborn serum promotes thermogenic competence, and the use of fetal serum in brown adipocyte cultures (as is often routine) counteracts adequate differentiation. Agents that counteract this inhibition may therefore confoundingly be ascribed genuine thermogenic competence-inducing properties.

Extra heat for defense of body temperature can be obtained from shivering or nonshivering thermogenesis. Nonshivering thermogenesis is a facultative (i.e., only occurring when needed) and adaptive (i.e., being augmented when the demand is chronically higher) process that, in mammals, is the result of the activity of uncoupling protein-1 (UCP1) in brown and brownish adipose tissues; no other quantitatively significant mechanism that fulfills the above criteria has been established. Measurement of heat production is generally indirect, based on oxygen consumption. Heat from brown adipose tissue is generated in mammals adapted to cold, in mammalian neonates, and in mammalian hibernators during arousal; brown adipose tissue may also be active in obese mammals and thus partially protect against further obesity. UCP1 is innately inhibited by cytosolic adenosine triphosphate (ATP) and is likely activated by fatty acids released from triglycerides within the cells; this lipolysis is stimulated by norepinephrine released from the sympathetic nerves innervating the tissue. For prolonged thermogenesis, substrate is delivered by the circulation as chylomicrons, lipoproteins, fatty acids, and glucose. The proton gradient over the mitochondrial membrane created by the respiratory chain is dispersed through the activity of UCP1; brown adipose tissue is nearly devoid of ATP synthase (as compared to respiratory chain activity). UCP1 developed likely at the dawn of mammalian evolution; most mammalian species still retain functional UCP1. Other members of the uncoupling protein family cannot uncouple. Both newborn and adult humans possess active brown adipose tissue but the significance of the tissue for adult human metabolism is not established., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The significance of diet-induced thermogenesis (DIT) for metabolic control is still debated. Although obesogenic diets recruit UCP1 and adrenergically inducible thermogenesis, and although the absence of UCP1 may promote the development of obesity, no actual UCP1-related thermogenesis identifiable as diet-induced thermogenesis has to date been unambiguously demonstrated. Examining mice living at thermoneutrality, we have identified a process of facultative (directly elicited by acute eating), adaptive (magnitude develops over weeks on an obesogenic diet), and fully UCP1-dependent thermogenesis. We found no evidence for UCP1-independent diet-induced thermogenesis. The thermogenesis was proportional to the total amount of UCP1 protein in brown adipose tissue and was not dependent on any contribution of UCP1 in brite/beige adipose tissue, since no UCP1 protein was found there under these conditions. Total UCP1 protein amount developed proportionally to total body fat content. The physiological messenger linking obesity level and acute eating to increased thermogenesis is not known. Thus UCP1-dependent diet-induced thermogenesis limits obesity development during exposure to obesogenic diets but does not prevent obesity as such., (Copyright © 2017 the American Physiological Society.)

Adaptive thermogenesis is the process of heat generation in response to cold stimulation. It is under the control of the sympathetic nervous system, whose chief effector is the catecholamine norepinephrine (NE). NE enhances thermogenesis through β3-adrenergic receptors to activate brown adipose tissue and by 'browning' white adipose tissue. Recent studies have reported that alternative activation of macrophages in response to interleukin (IL)-4 stimulation induces the expression of tyrosine hydroxylase (TH), a key enzyme in the catecholamine synthesis pathway, and that this activation provides an alternative source of locally produced catecholamines during the thermogenic process. Here we report that the deletion of Th in hematopoietic cells of adult mice neither alters energy expenditure upon cold exposure nor reduces browning in inguinal adipose tissue. Bone marrow-derived macrophages did not release NE in response to stimulation with IL-4, and conditioned media from IL-4-stimulated macrophages failed to induce expression of thermogenic genes, such as uncoupling protein 1 (Ucp1), in adipocytes cultured with the conditioned media. Furthermore, chronic treatment with IL-4 failed to increase energy expenditure in wild-type, Ucp1 -/- and interleukin-4 receptor-α double-negative (Il4ra -/- ) mice. In agreement with these findings, adipose-tissue-resident macrophages did not express TH. Thus, we conclude that alternatively activated macrophages do not synthesize relevant amounts of catecholamines, and hence, are not likely to have a direct role in adipocyte metabolism or adaptive thermogenesis.

The possibility that brown adipose tissue thermogenesis can be recruited in order to combat the development of obesity has led to a high interest in the identification of "browning agents", i.e. agents that increase the amount and activity of UCP1 in brown and brite/beige adipose tissues. However, functional analysis of the browning process yields confusingly different results when the analysis is performed in one of two alternative steps. Thus, in one of the steps, using cold acclimation as a potent model browning agent, we find that if the browning process is followed in mice initially housed at 21 °C (the most common procedure), there is only weak molecular evidence for increases in UCP1 gene expression or UCP1 protein abundance in classical brown adipose tissue; however, in brite/beige adipose depots, there are large increases, apparently associating functional browning with events only in the brite/beige tissues. Contrastingly, in another step, if the process is followed starting with mice initially housed at 30 °C (thermoneutrality for mice, thus similar to normal human conditions), large increases in UCP1 gene expression and UCP1 protein abundance are observed in the classical brown adipose tissue depots; there is then practically no observable UCP1 gene expression in brite/beige tissues. This apparent conundrum can be resolved when it is realized that the classical brown adipose tissue at 21 °C is already essentially fully differentiated and thus expands extensively through proliferation upon further browning induction, rather than by further enhancing cellular differentiation. When the limiting factor for thermogenesis, i.e. the total amount of UCP1 protein per depot, is analyzed, classical brown adipose tissue is by far the predominant site for the browning process, irrespective of which of the two steps is analyzed. There are to date no published data demonstrating that alternative browning agents would selectively promote brite/beige tissues versus classical brown tissue to a higher degree than does cold acclimation. Thus, to restrict investigations to examine adipose tissue depots where only a limited part of the adaptation process occurs (i.e. the brite/beige tissues) and to use initial conditions different from the thermoneutrality normally experienced by adult humans may seriously hamper the identification of therapeutically valid browning agents. The data presented here have therefore important implications for the analysis of the potential of browning agents and the nature of human brown adipose tissue., (Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.)

Cidea is a gene highly expressed in thermogenesis-competent (UCP1-containing) adipose cells, both brown and brite/beige. Here, we initially demonstrate a remarkable adipose-depot specific regulation of Cidea expression. In classical brown fat, Cidea mRNA is expressed continuously and invariably, irrespective of tissue recruitment. However, Cidea protein levels are regulated posttranscriptionally, being conspicuously induced in the thermogenically recruited state. In contrast, in brite fat, Cidea protein levels are regulated at the transcriptional level, and Cidea mRNA and protein levels are proportional to tissue "briteness." Although routinely followed as a thermogenic molecular marker, Cidea function is not clarified. Here, we employed a gain-of-function approach to examine a possible role of Cidea in the regulation of thermogenesis. We utilized transgenic aP2-hCidea mice that overexpress human Cidea in all adipose tissues. We demonstrate that UCP1 activity is markedly suppressed in brown-fat mitochondria isolated from aP2-hCidea mice. However, mitochondrial UCP1 protein levels were identical in wild-type and transgenic mice. This implies a regulatory effect of Cidea on UCP1 activity, but as we demonstrate that Cidea itself is not localized to mitochondria, we propose an indirect inhibitory effect. The Cidea-induced inhibition of UCP1 activity (observed in isolated mitochondria) is physiologically relevant since the mice, through an appropriate homeostatic compensatory mechanism, increased the total amount of UCP1 in the tissue to exactly match the diminished thermogenic capacity of the UCP1 protein and retain unaltered nonshivering thermogenic capacity. Thus, we verified Cidea as being a marker of thermogenesis-competent adipose tissues, but we conclude that Cidea, unexpectedly, functions molecularly as an indirect inhibitor of thermogenesis., (Copyright © 2017 the American Physiological Society.)

Leptin has been believed to exert its weight-reducing action not only by inducing hypophagia but also by increasing energy expenditure/thermogenesis. Leptin-deficient ob/ob mice have correspondingly been thought to be thermogenically limited and to show hypothermia, mainly due to atrophied brown adipose tissue (BAT). In contrast to these established views, we found that BAT is fully functional and that leptin treatment did not increase thermogenesis in wild-type or in ob/ob mice. Rather, ob/ob mice showed a decreased but defended body temperature (i.e., were anapyrexic, not hypothermic) that was normalized to wild-type levels after leptin treatment. This was not accompanied by increased energy expenditure or BAT recruitment but, instead, was mediated by decreased tail heat loss. The weight-reducing hypophagic effects of leptin are, therefore, not augmented through a thermogenic effect of leptin; leptin is, however, pyrexic, i.e., it alters centrally regulated thresholds of thermoregulatory mechanisms, in parallel to effects of other cytokines., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Brown adipose tissue (BAT) thermogenesis relies on blood flow to be supplied with nutrients and oxygen and for the distribution of the generated heat to the rest of the body. Therefore, it is fundamental to understand the mechanisms by which blood flow is regulated and its relation to thermogenesis. Here, we present high-resolution laser-Doppler imaging (HR-LDR) as a novel method for noninvasive in vivo measurement of BAT blood flow in mice. Using HR-LDR, we found that norepinephrine stimulation increases BAT blood flow in a dose-dependent manner and that this response is profoundly modulated by environmental temperature acclimation. Surprisingly, we found that mice lacking uncoupling protein 1 (UCP1) have fully preserved BAT blood flow response to norepinephrine despite failing to perform thermogenesis. BAT blood flow was not directly correlated to systemic glycemia, but glucose injections could transiently increase tissue perfusion. Inguinal white adipose tissue, also known as a brite/beige adipose tissue, was also sensitive to cold acclimation and similarly increased blood flow in response to norepinephrine. In conclusion, using a novel noninvasive method to detect BAT perfusion, we demonstrate that adrenergically stimulated BAT blood flow is qualitatively and quantitatively fully independent of thermogenesis, and therefore, it is not a reliable parameter for the estimation of BAT activation and heat generation., (Copyright © 2015 the American Physiological Society.)

The phenomenon of white fat "browning," in which certain white adipose tissue depots significantly increase gene expression for the uncoupling protein UCP1 and thus supposedly acquire thermogenic, fat-burning properties, has attracted considerable attention. Because the mRNA increases are from very low initial levels, the metabolic relevance of the change is unclear: is the UCP1 protein thermogenically competent in these brite/beige-fat mitochondria? We found that, in mitochondria isolated from the inguinal "white" adipose depot of cold-acclimated mice, UCP1 protein levels almost reached those in brown-fat mitochondria. The UCP1 was thermogenically functional, in that these mitochondria exhibited UCP1-dependent thermogenesis with lipid or carbohydrate substrates with canonical guanosine diphosphate (GDP) sensitivity and loss of thermogenesis in UCP1 knockout (KO) mice. Obesogenic mouse strains had a lower thermogenic potential than obesity-resistant strains. The thermogenic density (UCP1-dependent oxygen consumption per g tissue) of inguinal white adipose tissue was maximally one-fifth of interscapular brown adipose tissue, and the total quantitative contribution of all inguinal mitochondria was maximally one-third of all interscapular brown-fat mitochondria, indicating that the classical brown adipose tissue depots would still predominate in thermogenesis., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Because of the possible role of brown adipose tissue and UCP1 in metabolic regulation, even in adult humans, there is presently considerable interest in quantifying, from in-vitro data, the thermogenic capacities of brown and brite/beige adipose tissues. An important issue is therefore to establish which parameters are the most adequate for this. A particularly important issue is the relevance of UCP1 mRNA levels as estimates of the degree of recruitment and of the thermogenic capacity resulting from differences in physiological conditions and from experimental manipulations. By solely following UCP1 mRNA levels in brown adipose tissue, the conclusion would be made that the tissue's highest activation occurs after only 6h in the cold and then successively decreases to being only some 50% elevated after 1month in the cold. However, measurement of total UCP1 protein levels per depot ("mouse") reveals that the maximal thermogenic capacity estimated in this way is reached first after 1month but represents an approx. 10-fold increase in thermogenic capacity. Since this in-vitro measure correlates quantitatively and temporally with the acquisition of nonshivering thermogenesis, this must be considered the most physiologically relevant parameter. Similarly, observations that cold acclimation barely increases UCP1 mRNA levels in classical brown adipose tissue but leads to a 200-fold increase in UCP1 mRNA levels in brite/beige adipose tissue depots may overemphasise the physiological significance of these depots, as the high fold-increases are due to very low initial levels, and the UCP1 mRNA levels reached are at least an order of magnitude lower than in brown adipose tissue; furthermore, based on total UCP1 protein amounts, the brite/beige depots attain only about 10% of the thermogenic capacity of the classical brown adipose tissue depots. Consequently, inadequate conclusions may be reached if UCP1 mRNA levels are used as a proxy for the metabolic significance of recruited versus non-recruited brown adipose tissue and for estimating the metabolic significance of brown versus brite/beige adipose tissues. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Thermogenesis in brown adipose tissue (BAT) is fundamental to energy balance and is also relevant for humans. Bone morphogenetic proteins (BMPs) regulate adipogenesis, and, here, we describe a role for BMP8B in the direct regulation of thermogenesis. BMP8B is induced by nutritional and thermogenic factors in mature BAT, increasing the response to noradrenaline through enhanced p38MAPK/CREB signaling and increased lipase activity. Bmp8b(-/-) mice exhibit impaired thermogenesis and reduced metabolic rate, causing weight gain despite hypophagia. BMP8B is also expressed in the hypothalamus, and Bmp8b(-/-) mice display altered neuropeptide levels and reduced phosphorylation of AMP-activated protein kinase (AMPK), indicating an anorexigenic state. Central BMP8B treatment increased sympathetic activation of BAT, dependent on the status of AMPK in key hypothalamic nuclei. Our results indicate that BMP8B is a thermogenic protein that regulates energy balance in partnership with hypothalamic AMPK. BMP8B may offer a mechanism to specifically increase energy dissipation by BAT., (Copyright © 2012 Elsevier Inc. All rights reserved.)

That adult humans possess brown fat is now accepted - but is the brown fat metabolically active? Does human brown fat actually combust fat to release heat? In this issue of the JCI, Ouellet et al. demonstrate that metabolism in brown fat really is increased when adult humans are exposed to cold. This boosts the possibility that calorie combustion in brown fat may be of significance for our metabolism and, correspondingly, that the absence of brown fat may increase our proneness to obesity - provided that brown fat becomes activated not only by cold but also through food-related stimuli.

Alterations in nonshivering thermogenesis are presently discussed as being both potentially causative of and able to counteract obesity. However, the necessity for mammals to defend their body temperature means that the ambient temperature profoundly affects the outcome and interpretation of metabolic experiments. An adequate understanding and assessment of nonshivering thermogenesis is therefore paramount for metabolic studies. Classical nonshivering thermogenesis is facultative, i.e. it is only activated when an animal acutely requires extra heat (switched on in minutes), and adaptive, i.e. it takes weeks for an increase in capacity to develop. Nonshivering thermogenesis is fully due to brown adipose tissue activity; adaptation corresponds to the recruitment of this tissue. Diet-induced thermogenesis is probably also facultative and adaptive and due to brown adipose tissue activity. Although all mammals respond to injected/infused norepinephrine (noradrenaline) with an increase in metabolism, in non-adapted mammals this increase mainly represents the response of organs not involved in nonshivering thermogenesis; only the increase after adaptation represents nonshivering thermogenesis. Thermogenesis (metabolism) should be expressed per animal, and not per body mass [not even to any power (0.75 or 0.66)]. A 'cold tolerance test' does not examine nonshivering thermogenesis capacity; rather it tests shivering capacity and endurance. For mice, normal animal house temperatures are markedly below thermoneutrality, and the mice therefore have a metabolic rate and food consumption about 1.5 times higher than their intrinsic requirements. Housing and examining mice at normal house temperatures carries a high risk of identifying false positives for intrinsic metabolic changes; in particular, mutations/treatments that affect the animal's insulation (fur, skin) may lead to such problems. Correspondingly, true alterations in intrinsic metabolic rate remain undetected when metabolism is examined at temperatures below thermoneutrality. Thus, experiments with animals kept and examined at thermoneutrality are likely to yield an improved possibility of identifying agents and genes important for human energy balance.

Only with the development of the uncoupling protein 1 (UCP1)-ablated mouse has it become possible to strictly delineate the physiological significance of the thermogenic capacity of brown adipose tissue. Considering the presence of active brown adipose tissue in adult humans, these insights may have direct human implications. In addition to classical nonshivering thermogenesis, all adaptive adrenergic thermogeneses, including diet-induced thermogenesis, is fully dependent on brown adipocyte activity. Any weight-reducing effect of β(3)-adrenergic agonists is fully dependent on UCP1 activity, as is any weight-reducing effect of leptin (in excess of its effect on reduction of food intake). Consequently, in the absence of the thermogenic activity of brown adipose tissue, obesity develops spontaneously. The ability of brown adipose tissue to contribute to glucose disposal is also mainly related to thermogenic activity. However, basal metabolic rate, cold-induced thermogenesis, acute cold tolerance, fevers, nonadaptive adrenergic thermogenesis and processes such as angiogenesis in brown adipose tissue itself are not dependent on UCP1 activity. Whereas it is likely that these conclusions are also qualitatively valid for adult humans, the quantitative significance of brown adipose tissue for human metabolism--and the metabolic consequences for a single individual possessing more or less brown adipose tissue--awaits clarification.

Caveolin-1 (Cav1)-ablated mice display impaired lipolysis in white adipose tissue. They also seem to have an impairment in brown adipose tissue function, implying that Cav1-ablated mice could encounter problems in surviving longer periods in cold temperatures. To investigate this, Cav1-ablated mice and wild-type mice were transferred to cold temperatures for extended periods of time, and parameters related to metabolism and thermogenesis were investigated. Unexpectedly, the Cav1-ablated mice survived in the cold. There were no differences between Cav1-ablated and wild-type mice with regard to food intake, in behavior related to shivering, or in body temperature. The Cav1-ablated mice had a halved total fat content independently of acclimation temperature. There was no difference in brown adipose tissue uncoupling protein-1 (UCP1) protein amount, and isolated brown fat mitochondria were thermogenically competent but displayed 30% higher thermogenic capacity. However, the beta(3)-adrenergic receptor amount was reduced by about one-third in the Cav1-ablated mice at all acclimation temperatures. Principally in accordance with this, a higher than standard dose of norepinephrine was needed to obtain full norepinephrine-induced thermogenesis in the Cav1-ablated mice; the higher dose was also needed for the Cav1-ablated mice to be able to utilize fat as a substrate for thermogenesis. In conclusion, the ablation of Cav1 impairs brown adipose tissue function by a desensitization of the adrenergic response; however, the desensitization is not evident in the animal as it is overcome physiologically, and Cav1-ablated mice can therefore survive in prolonged cold by nonshivering thermogenesis.

That adult humans possess active brown adipose tissue potentially leads to a paradigm shift in the understanding of human metabolism and of obesity. Adaptive adrenergic thermogenesis in humans represents brown adipose tissue activity, the absence of which may contribute to middle-age obesity., (2010 Elsevier Inc. All rights reserved.)

The recent insight that brown adipocytes and muscle cells share a common origin and in this respect are distinct from white adipocytes has spurred questions concerning the origin and molecular characteristics of the UCP1-expressing cells observed in classic white adipose tissue depots under certain physiological or pharmacological conditions. Examining precursors from the purest white adipose tissue depot (epididymal), we report here that chronic treatment with the peroxisome proliferator-activated receptor gamma agonist rosiglitazone promotes not only the expression of PGC-1alpha and mitochondriogenesis in these cells but also a norepinephrine-augmentable UCP1 gene expression in a significant subset of the cells, providing these cells with a genuine thermogenic capacity. However, although functional thermogenic genes are expressed, the cells are devoid of transcripts for the novel transcription factors now associated with classic brown adipocytes (Zic1, Lhx8, Meox2, and characteristically PRDM16) or for myocyte-associated genes (myogenin and myomirs (muscle-specific microRNAs)) and retain white fat characteristics such as Hoxc9 expression. Co-culture experiments verify that the UCP1-expressing cells are not proliferating classic brown adipocytes (adipomyocytes), and these cells therefore constitute a subset of adipocytes ("brite" adipocytes) with a developmental origin and molecular characteristics distinguishing them as a separate class of cells.

According to the adipostat hypothesis for body-weight control, alterations in body weight should always be compensated by adequate alterations in food intake and thermogenesis. Thus, increased thermogenesis should not be able to counteract obesity because food intake would be increased. However evidence is presented here that thermogenesis in different forms (through artificial uncouplers, exercise, cold exposure) may counteract obesity and is not always fully compensated by increased food intake. Correspondingly, a decreased capacity for metaboloregulatory thermogenesis (i.e. non-functional brown adipose tissue) may in itself lead to obesity. This is evident in mice and may be valid for human subjects, as a substantial proportion of adults possess brown adipose tissue, and those with less or without brown adipose tissue would seem to be more prone to obesity. Thus, increased thermogenesis may counteract obesity, without dietary intervention.

As original studies of UCP1-ablated mice failed to demonstrate an obesogenic effect, alternative mechanisms for adaptive adrenergic thermogenesis have been sought. However, we demonstrate here that in C57Bl6 mice exempt from thermal stress (i.e., kept at thermoneutrality), UCP1 ablation in itself induced obesity, even in mice fed control diet, and vastly augmented diet-induced obesity (high-fat diet); i.e., the mice exhibited increased metabolic efficiency. In wild-type mice, high-fat diet increased norepinephrine-induced thermogenesis; i.e., diet-induced thermogenesis was observed, but no such effect was observed in UCP1-ablated mice, demonstrating that diet-induced thermogenesis fully emanates from UCP1 activity. We conclude that ambient temperature is qualitatively determinative for the outcome of metabolic studies, that no other protein and no other mechanism can substitute for UCP1 in mediating diet-induced adrenergic thermogenesis, and that UCP1 activity can be determinative for obesity development in mice and possibly in humans.

When acutely exposed to a cold environment, mammals shiver to generate heat. During prolonged cold exposure, shivering is replaced by adaptive adrenergic nonshivering thermogenesis with increased heat production in brown adipose tissue due to activation of uncoupling protein-1 (UCP1). This cold acclimation is associated with chronically increased sympathetic stimulation of skeletal muscle, which may increase the sarcoplasmic reticulum (SR) Ca(2+) leak via destabilized ryanodine receptor 1 (RyR1) channel complexes. Here, we use genetically engineered UCP1-deficient (UCP1-KO) mice that rely completely on shivering in the cold. We examine soleus muscle, which participates in shivering, and flexor digitorum brevis (FDB) muscle, a distal and superficial muscle that does not shiver. Soleus muscles of cold-acclimated UCP1-KO mice exhibited severe RyR1 PKA hyperphosphorylation and calstabin1 depletion, as well as markedly decreased SR Ca(2+) release and force during contractions. In stark contrast, the RyR1 channel complexes were little affected, and Ca(2+) and force were not decreased in FDB muscles of cold-acclimated UCP1-KO mice. These results indicate that activation of UCP1-mediated heat production in brown adipose tissue during cold exposure reduces the necessity for shivering and thus prevents the development of severe dysfunction in shivering muscles.

Most physiologically induced examples of recruitment of brown adipose tissue (BAT) occur as a consequence of chronic sympathetic stimulation (norepinephrine release within the tissue). However, in some physiological contexts (e.g., prenatal and prehibernation recruitment), this pathway is functionally contraindicated. Thus a nonsympathetically mediated mechanism of BAT recruitment must exist. Here we have tested whether a PPARgamma activation pathway could competently recruit BAT, independently of sympathetic stimulation. We continuously treated primary cultures of mouse brown (pre)adipocytes with the potent peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist rosiglitazone. In rosiglitazone-treated cultures, morphological signs of adipose differentiation and expression levels of the general adipogenic marker aP2 were manifested much earlier than in control cultures. Importantly, in the presence of the PPARgamma agonist the brown adipocyte phenotype was significantly enhanced: UCP1 was expressed even in the absence of norepinephrine, and PPARalpha expression and norepinephrine-induced PGC-1alpha mRNA levels were significantly increased. However, the augmented levels of PPARalpha could not explain the brown-fat promoting effect of rosiglitazone, as this effect was still evident in PPARalpha-null cells. In continuously rosiglitazone-treated brown adipocytes, mitochondriogenesis, an essential part of BAT recruitment, was significantly enhanced. Most importantly, these mitochondria were capable of thermogenesis, as rosiglitazone-treated brown adipocytes responded to the addition of norepinephrine with a large increase in oxygen consumption. This thermogenic response was not observable in rosiglitazone-treated brown adipocytes originating from UCP1-ablated mice; hence, it was UCP1 dependent. Thus the PPARgamma pathway represents an alternative, potent, and fully competent mechanism for BAT recruitment, which may be the cellular explanation for the enigmatic recruitment in prehibernation and prenatal states.

Brown and white adipose tissues in mammals have a number of similar properties, such as lipid storage and adipokine production, but also distinctive properties. The energy-storing white adipose tissue has few mitochondria and low oxidative capacity. The heat-producing brown adipose tissue has a high density of mitochondria and high oxidative capacity. Mitochondrial function can be investigated in cells and organelles isolated from both brown and white adipose tissues. This chapter describes methods for successful isolation of suitable preparations of adipose tissues and their subsequent use. Questions concerning thermogenic capacity of the tissues, their potential influence on whole body metabolism, and specific properties of the mitochondria and their mode of function may be addressed using these methods.

Mice with a dominant-negative peroxisome proliferator-activated receptor gamma (PPARgamma) mutation (P465L) unexpectedly had normal amounts of adipose tissue. Here, we investigate the adipose tissue of the PPARgamma P465L mouse in detail. Microscopic analysis of interscapular adipose tissue of P465L PPARgamma mice revealed brown adipocytes with larger unilocular lipid droplets, indicative of reduced thermogenic capacity. Under conditions of cold exposure, the brown adipose tissue of the PPARgamma P465L mice was less active, a fact reflected in decreased uncoupling protein 1 levels. Analysis of the white adipocytes confirmed their normal cytoarchitecture and development, yet classical white adipose depots of the P465L PPARgamma mice had a striking reduction in brown adipocyte recruitment, a finding supported by reduced expression of UCP1 in the perigonadal adipose depot. Taken together, these data suggest that whole animal impairment of PPARgamma alters the cellular composition of the adipose organ to a more "white" adipose phenotype. Physiologically, this impairment in brown adipocyte recruitment is associated with decreased nonshivering thermogenic capacity after cold acclimation as revealed by norepinephrine responsiveness. Our results indicate that maintenance of oxidative brown-like adipose tissue is more dependent on PPARgamma function for development than white adipose tissue, an observation that may be relevant when considering PPARgamma-dependent strategies for the treatment of obesity.

The transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator-1beta (PGC-1beta) has been implicated in important metabolic processes. A mouse lacking PGC-1beta (PGC1betaKO) was generated and phenotyped using physiological, molecular, and bioinformatic approaches. PGC1betaKO mice are generally viable and metabolically healthy. Using systems biology, we identified a general defect in the expression of genes involved in mitochondrial function and, specifically, the electron transport chain. This defect correlated with reduced mitochondrial volume fraction in soleus muscle and heart, but not brown adipose tissue (BAT). Under ambient temperature conditions, PGC-1beta ablation was partially compensated by up-regulation of PGC-1alpha in BAT and white adipose tissue (WAT) that lead to increased thermogenesis, reduced body weight, and reduced fat mass. Despite their decreased fat mass, PGC1betaKO mice had hypertrophic adipocytes in WAT. The thermogenic role of PGC-1beta was identified in thermoneutral and cold-adapted conditions by inadequate responses to norepinephrine injection. Furthermore, PGC1betaKO hearts showed a blunted chronotropic response to dobutamine stimulation, and isolated soleus muscle fibres from PGC1betaKO mice have impaired mitochondrial function. Lack of PGC-1beta also impaired hepatic lipid metabolism in response to acute high fat dietary loads, resulting in hepatic steatosis and reduced lipoprotein-associated triglyceride and cholesterol content. Altogether, our data suggest that PGC-1beta plays a general role in controlling basal mitochondrial function and also participates in tissue-specific adaptive responses during metabolic stress.

Participation of brown adipose tissue [through the action of the uncoupling protein-1 (UCP1)] in adaptive adrenergic nonshivering thermogenesis is recognized, but the existence of a response to adrenergic stimulation in UCP1-ablated mice implies that a mechanism for an alternative adaptive adrenergic thermogenesis may exist. Here, we have used UCP1-ablated mice to examine the existence of an alternative adaptive adrenergic nonshivering thermogenesis, examined as the oxygen consumption response to systemically injected norepinephrine into anesthetized or conscious mice acclimated to different temperatures. We confirm that UCP1-dependent adrenergic nonshivering thermogenesis is adaptive, but we demonstrate that the adrenergic UCP1-independent thermogenesis is not recruitable by cold acclimation. Thus, at least in the mouse, no other proteins or enzymatic pathways exist that can participate in or with time take over the UCP1 mediation of adaptive adrenergic nonshivering thermogenesis, even in the total absence of UCP1. UCP1 is thus the only protein capable of mediating cold acclimation-recruited adaptive adrenergic nonshivering thermogenesis.

Volatile anesthetics such as halothane efficiently inhibit nonshivering thermogenesis as well as the cellular manifestation of that phenomenon: norepinephrine-induced respiration in brown adipocytes. To identify the molecular site(s) of action of such anesthetics, we have examined the effect of halothane on the sequential intracellular steps from the interaction of norepinephrine with isolated brown adipocytes to the stimulation of mitochondrial respiration (=thermogenesis). We did not identify an inhibition at the level of the adrenergic receptors, but a first site of inhibition was identified as the generation of cAMP by adenylyl cyclase; this led to inhibition of norepinephrine-induced expression of the uncoupling protein-1 (UCP1) gene and reduced norepinephrine-induced lipolysis as secondary effects. Although an inhibition of lipolysis in itself would inhibit thermogenesis, circumvention of this inhibition revealed that a second, postlipolytic, site of inhibition existed: halothane also inhibited the stimulatory effect of exogenous fatty acids on cellular respiration. This inhibition was independent of the presence of UCP1 in the mitochondria of the cells and was thus not directly on the thermogenic uncoupling mechanism. Since not only fatty acid oxidation but also pyruvate oxidation were inhibited by halothane in isolated mitochondria, whereas glycerol-3-phosphate oxidation was not, the second site of action of halothane, evident when cyclase/lipolytic inhibition was circumvented, was located to the respiratory chain, complex I. The results thus explain the inhibition of nonshivering thermogenesis by identifying two sites of action of halothane in brown adipocytes. In addition, the results may open for new formulations of the molecular background to anesthesia.

We have examined the metabolic role of hormone-binding nuclear thyroid hormone receptors (TRs). Mice devoid of all hormone-binding TRs [TR alpha 1(-/-)beta(-/-) (TR-ablated mice)] had slightly decreased body temperature and much decreased basal metabolic rate, were still able to markedly increase metabolic rate in the cold, but were cold intolerant due to inadequate total heat production at low temperatures. A standard norepinephrine test showed that adrenergically induced thermogenesis could not be activated normally in the TR-ablated mice. This was not due to inadequate recruitment of brown adipose tissue, nor to the absence, decreased recruitment or dysfunction of the uncoupling protein-1. However, isolated brown fat cells were 10-fold desensitized, explaining the lack of response to standard adrenergic stimuli; cell culture experiments demonstrated that this desensitization was not an innate effect. Thus, the cold intolerance was probably not due to inadequate sympathetically induced nonshivering thermogenesis. Additionally, the results indicated that no metabolic effects of thyroid hormones could become manifest in the absence of nuclear TRs, that ligand-bound TRs were needed for euthermia and eumetabolism, but that TRs per se were not required for brown adipose tissue recruitment and uncoupling protein-1 gene expression.

The function of brown adipose tissue is to transfer energy from food into heat; physiologically, both the heat produced and the resulting decrease in metabolic efficiency can be of significance. Both the acute activity of the tissue, i.e., the heat production, and the recruitment process in the tissue (that results in a higher thermogenic capacity) are under the control of norepinephrine released from sympathetic nerves. In thermoregulatory thermogenesis, brown adipose tissue is essential for classical nonshivering thermogenesis (this phenomenon does not exist in the absence of functional brown adipose tissue), as well as for the cold acclimation-recruited norepinephrine-induced thermogenesis. Heat production from brown adipose tissue is activated whenever the organism is in need of extra heat, e.g., postnatally, during entry into a febrile state, and during arousal from hibernation, and the rate of thermogenesis is centrally controlled via a pathway initiated in the hypothalamus. Feeding as such also results in activation of brown adipose tissue; a series of diets, apparently all characterized by being low in protein, result in a leptin-dependent recruitment of the tissue; this metaboloregulatory thermogenesis is also under hypothalamic control. When the tissue is active, high amounts of lipids and glucose are combusted in the tissue. The development of brown adipose tissue with its characteristic protein, uncoupling protein-1 (UCP1), was probably determinative for the evolutionary success of mammals, as its thermogenesis enhances neonatal survival and allows for active life even in cold surroundings.

Background: This investigation examined the possibility that the inhibitory effect of halothane on nonshivering thermogenesis (heat production) in brown adipocytes is not a universal effect of all anesthetic agents but related to the type of anesthetic., Methods: Brown adipocytes from hamster were isolated with a collagenase digestion method and incubated with anesthetic agents. The rate of oxygen consumption was measured with an oxygen electrode. The effect of clinically relevant (and higher) doses of anesthetics of different classes on basal and norepinephrine-induced thermogenesis (oxygen consumption) was tested., Results: Two distinct groups of anesthetics could be distinguished: thermogenesis inhibitors and noninhibitors. Thermogenesis inhibitors include volatile anesthetics such as halothane (IC(50), 1.1 mm), ether (IC(50), 20 mm), and chloroform (IC(50), 2.2 mm) (nominal concentrations), but also tribromoethanol (IC(50), 0.6 mm), all inducing inhibition of norepinephrine-induced thermogenesis without affecting the EC for norepinephrine. Thermogenesis noninhibitors include the nonvolatile anesthetics pentobarbital, propofol, ketamine, and urethane, the inhalation anesthetic nitrous oxide, and, notably, also the volatile nonanesthetics (nonimmobilizers) 1,2-dichlorohexafluorocyclobutane and 2,3-dichlorooctafluorobutane; none of these compounds had any effect on norepinephrine-induced thermogenesis at any concentration tested., Conclusions: There are two distinct classes of anesthetics with regard to effects on thermogenesis, thermogenesis inhibitors and thermogenesis noninhibitors. The results are important for the interpretation of studies in thermal biology in general; specifically, they indicate that conclusions concerning regulation of nonshivering thermogenesis during anesthesia depend on the type of anesthetic used. Of clinical importance is that the volatile anesthetics are inhibitory for nonshivering thermogenesis and thus for an alternative heat production when myorelaxants prevent shivering. As the distinction between thermogenesis inhibitors and thermogenesis noninhibitors corresponds to the distinction between volatile and nonvolatile anesthetics, it may be related to the mode of action of the volatile anesthetics.

The uniqueness of UCP1 (as compared to UCP2/UCP3) is evident from expression analysis and ablation studies. UCP1 expression is positively correlated with metabolic inefficiency, being increased by cold acclimation (in adults or perinatally) and overfeeding, and reduced in fasting and genetic obesity. Such a simple relationship is not observable for UCP2/UCP3. Studies with UCP1-ablated animals substantiate the unique role of UCP1: the phenomenon of adaptive adrenergic non-shivering thermogenesis in the intact animal is fully dependent on the presence of UCP1, and so is any kind of cold acclimation-recruited non-shivering thermogenesis; thus UCP2/UCP3 (or any other proteins or metabolic processes) cannot substitute for UCP1 physiologically, irrespective of their demonstrated ability to show uncoupling in reconstituted systems or when ectopically expressed. Norepinephrine-induced thermogenesis in brown-fat cells is absolutely dependent on UCP1, as is the uncoupled state and the recoupling by purine nucleotides in isolated brown-fat mitochondria. Although very high UCP2/UCP3 mRNA levels are observed in brown adipose tissue of UCP1-ablated mice, there is no indication that the isolated brown-fat mitochondria are uncoupled; thus, high expression of UCP2/UCP3 does not necessarily confer to the mitochondria of a tissue a propensity for being innately uncoupled. Whereas the thermogenic effect of fatty acids in brown-fat cells is fully UCP1-dependent, this is not the case in brown-fat mitochondria; this adds complexity to the issues concerning the mechanisms of UCP1 function and the pathway from beta(3)-adrenoceptor stimulation to UCP1 activation and thermogenesis. In addition to amino acid sequences conserved in all UCPs as part of the tripartite structure, all UCPs contain certain residues associated with nucleotide binding. However, conserved amongst all UCP1s so far sequenced, and without parallel in all UCP2/UCP3, are two sequences: 144SHLHGIKP and the C-terminal sequence RQTVDC(A/T)T; these sequences may therefore be essential for the unique thermogenic function of UCP1. The level of UCP1 in the organism is basically regulated at the transcriptional level (physiologically probably mainly through the beta(3)-adrenoceptor/CREB pathway), with influences from UCP1 mRNA stability and from the delay caused by translation. It is concluded that UCP1 is unique amongst the uncoupling proteins and is the only protein able to mediate adaptive non-shivering thermogenesis and the ensuing metabolic inefficiency.