UCP1-ablated Mice Research Articles

Uncoupling protein 1 and the capacity for nonshivering thermogenesis are components of the glucose homeostatic system.

Uncoupling protein 1 (Ucp1) provides nonshivering thermogenesis (NST) fueled by the dissipation of energy from macronutrients in brown and brite adipocytes. The availability of thermogenic fuels is facilitated by the uptake of extracellular glucose. This conjunction renders thermogenic adipocytes in brown and white adipose tissue (WAT) a potential target against obesity and glucose intolerance. We employed wild-type (WT) and Ucp1-ablated mice to elucidate this relationship. In three experiments of similar setup, Ucp1-ablated mice fed a high-fat diet (HFD) had either reduced or similar body mass gain, food intake, and metabolic efficiency compared with WT mice, challenging the hypothesized role of this protein in the development of diet-induced obesity. Despite the absence of increased body mass, oral glucose tolerance was robustly impaired in Ucp1-ablated mice in response to HFD. Postprandial glucose uptake was attenuated in brown adipose tissue but enhanced in subcutaneous WAT of Ucp1-ablated mice. These differences were explainable by expression of the insulin-responsive member 4 of the facilitated glucose transporter family and fully in line with the capacity for NST in these very tissues. Thus, the postprandial glucose uptake of adipose tissues serves as a surrogate measure for Ucp1-dependent and independent capacity for NST. Collectively, our findings corroborate Ucp1 as a modulator of adipose tissue glucose uptake and systemic glucose homeostasis but challenge its hypothesized causal effect on the development of obesity.

At thermoneutrality, acute thyroxine-induced thermogenesis and pyrexia are independent of UCP1

ObjectiveHyperthyroidism 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. MethodsWe 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. ResultsBoth 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. ConclusionsThese 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.

In the absence of UCP1-mediated diet-induced thermogenesis, obesity is augmented even in the obesity-resistant 129S mouse strain.

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.

Adaptive facultative diet-induced thermogenesis in wild-type but not in UCP1-ablated mice.

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.

Metabolically inert perfluorinated fatty acids directly activate uncoupling protein 1 in brown-fat mitochondria

The metabolically inert perfluorinated fatty acids perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) can display fatty acid-like activity in biological systems. The uncoupling protein 1 (UCP1) in brown adipose tissue is physiologically (re)activated by fatty acids, including octanoate. This leads to bioenergetically uncoupled energy dissipation (heat production, thermogenesis). We have examined here the possibility that PFOA/PFOS can directly (re)activate UCP1 in isolated mouse brown-fat mitochondria. In wild-type brown-fat mitochondria, PFOS and PFOA overcame GDP-inhibited thermogenesis, leading to increased oxygen consumption and dissipated membrane potential. The absence of this effect in brown-fat mitochondria from UCP1-ablated mice indicated that it occurred through activation of UCP1. A competitive type of inhibition by increased GDP concentrations indicated interaction with the same mechanistic site as that utilized by fatty acids. No effect was observed in heart mitochondria, i.e., in mitochondria without UCP1. The stimulatory effect of PFOA/PFOS was not secondary to non-specific mitochondrial membrane permeabilization or to ROS production. Thus, metabolic effects of perfluorinated fatty acids could include direct brown adipose tissue (UCP1) activation. The possibility that this may lead to unwarranted extra heat production and thus extra utilization of food resources, leading to decreased fitness in mammalian wildlife, is discussed, as well as possible negative effects in humans. However, a possibility to utilize PFOA-/PFOS-like substances for activating UCP1 therapeutically in obesity-prone humans may also be envisaged.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-015-1535-4) contains supplementary material, which is available to authorized users.

Increased fatigue resistance linked to Ca2+-stimulated mitochondrial biogenesis in muscle fibres of cold-acclimated mice

Mammals exposed to a cold environment initially generate heat by repetitive muscle activity (shivering). Shivering is successively replaced by the recruitment of uncoupling protein-1 (UCP1)-dependent heat production in brown adipose tissue. Interestingly, adaptations observed in skeletal muscles of cold-exposed animals are similar to those observed with endurance training. We hypothesized that increased myoplasmic free [Ca2+] ([Ca2+]i) is important for these adaptations. To test this hypothesis, experiments were performed on flexor digitorum brevis (FDB) muscles, which do not participate in the shivering response, of adult wild-type (WT) and UCP1-ablated (UCP1-KO) mice kept either at room temperature (24°C) or cold-acclimated (4°C) for 4-5 weeks. [Ca2+]i (measured with indo-1) and force were measured under control conditions and during fatigue induced by repeated tetanic stimulation in intact single fibres. The results show no differences between fibres from WT and UCP1-KO mice. However, muscle fibres from cold-acclimated mice showed significant increases in basal [Ca2+]i (∼50%), tetanic [Ca2+]i (∼40%), and sarcoplasmic reticulum (SR) Ca2+ leak (∼fourfold) as compared to fibres from room-temperature mice. Muscles of cold-acclimated mice showed increased expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and increased citrate synthase activity (reflecting increased mitochondrial content). Fibres of cold-acclimated mice were more fatigue resistant with higher tetanic [Ca2+]i and less force loss during fatiguing stimulation. In conclusion, cold exposure induces changes in FDB muscles similar to those observed with endurance training and we propose that increased [Ca2+]i is a key factor underlying these adaptations.

Torpor patterns, arousal rates, and temporal organization of torpor entry in wildtype and UCP1-ablated mice

In eutherian mammals, uncoupling protein 1 (UCP1) mediated non-shivering thermogenesis from brown adipose tissue (BAT) provides a mechanism through which arousal from torpor and hibernation is facilitated. In order to directly assess the magnitude by which the presence or absence of UCP1 affects torpor patterns, rewarming and arousal rates within one species we compared fasting induced torpor in wildtype (UCP1(+/+)) and UCP1-ablated mice (UCP(-/-)). Torpor was induced by depriving mice of food for up to 48 h and by a reduction of ambient temperature (T (a)) from 30 to 18°C at four different time points after 18, 24, 30 and 36 h of food deprivation. In most cases, torpor bouts occurred within 20 min after the switch in ambient temperature (30-18°C). Torpor bouts expressed during the light phase lasted 3-6 h while significantly longer bouts (up to 16 h) were observed when mice entered torpor during the dark phase. The degree of hypometabolism (5-22 ml h(-1)) and hypothermia (19.5-26.7°C) was comparable in wildtype and UCP1-ablated mice, and both genotypes were able to regain normothermia. In contrast to wildtype mice, UCP1-ablated mice did not display multiple torpor bouts per day and their peak rewarming rates from torpor were reduced by 50% (UCP1(+/+): 0.24 ± 0.08°C min(-1); UCP1(-/-): 0.12 ± 0.04°C min(-1)). UCP1-ablated mice therefore took significantly longer to rewarm from 25 to 32°C (39 vs. 70 min) and required 60% more energy for this process. Our results demonstrate the energetic benefit of functional BAT for rapid arousal from torpor. They also suggest that torpor entry and maintenance may be dependent on endogenous rhythms.

Cold tolerance of UCP1-ablated mice: A skeletal muscle mitochondria switch toward lipid oxidation with marked UCP3 up-regulation not associated with increased basal, fatty acid- or ROS-induced uncoupling or enhanced GDP effects

Mice lacking the thermogenic mitochondrial membrane protein UCP1 (uncoupling protein 1) - and thus all heat production from brown adipose tissue - can still adapt to a cold environment (4°C) if successively transferred to the cold. The mechanism behind this adaptation has not been clarified. To examine possible adaptive processes in the skeletal muscle, we isolated mitochondria from the hind limb muscles of cold-acclimated wild-type and UCP1(–/–) mice and examined their bioenergetic chracteristics. We observed a switch in metabolism, from carbohydrate towards lipid catabolism, and an increased total mitochondrial complement, with an increased total ATP production capacity. The UCP1(–/–) muscle mitochondria did not display a changed state-4 respiration rate (no uncoupling) and were less sensitive to the uncoupling effect of fatty acids than the wild-type mitochondria. The content of UCP3 was increased 3-4 fold, but despite this, endogenous superoxide could not invoke a higher proton leak, and the small inhibitory effect of GDP was unaltered, indicating that it was not mediated by UCP3. Double mutant mice (UCP1(–/–) plus superoxide dismutase 2-overexpression) were not more cold sensitive than UCP1(–/–), bringing into question an involvement of reactive oxygen species (ROS) in activation of any alternative thermogenic mechanism. We conclude that there is no evidence for an involvement of UCP3 in basal, fatty-acid- or superoxide-stimulated oxygen consumption or in GDP sensitivity. The adaptations observed did not imply any direct alternative process for nonshivering thermogenesis but the adaptations observed would be congruent with adaptation to chronically enhanced muscle activity caused by incessant shivering in these mice.

UCP1 Ablation Induces Obesity and Abolishes Diet-Induced Thermogenesis in Mice Exempt from Thermal Stress by Living at Thermoneutrality

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.

Thermogenically competent nonadrenergic recruitment in brown preadipocytes by a PPARγ agonist

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.

Cold-induced alterations of phospholipid fatty acyl composition in brown adipose tissue mitochondria are independent of uncoupling protein-1

The recruitment process induced by acclimation of mammals to cold includes a marked alteration in the acyl composition of the phospholipids of mitochondria from brown adipose tissue: increases in 18:0, 18:2(n-6), and 20:4(n-6) and decreases in 16:0, 16:1, 18:1, and 22:6(n-3). A basic question is whether these alterations are caused by changes in the concentration of uncoupling protein-1 (UCP1) or the thermogenesis it mediates-implying that they are secondary effects-or whether they are an integrated, independent part of the recruitment process. This question was addressed here using wild-type and UCP1-ablated C57BL/6 mice acclimated to 24 degrees C or 4 degrees C. In wild-type mice, the phospholipid fatty acyl composition of mitochondria from brown adipose tissue showed the changes in response to cold that were expected from observations in other species and strains. The changes were specific, as different changes occurred in skeletal muscle mitochondria. In UCP1-ablated mice, cold acclimation induced acyl alterations in brown adipose tissue that were qualitatively identical and quantitatively similar to those in wild-type mice. Therefore, neither the increased content of UCP1 nor mitochondrial uncoupling altered the effect of cold on acyl composition. Cold acclimation in wild-type mice had little effect on phospholipid acyl composition in muscle mitochondria, but cold-acclimation in UCP1-ablated mice caused significant alterations, probably due to sustained shivering. Thus, the alterations in brown adipose tissue phospholipid acyl composition are revealed to be an independent part of the recruitment process, and their functional significance for thermogenesis should be elucidated.

UCP1 is essential for adaptive adrenergic nonshivering thermogenesis

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.

Cold-induced expression of the VEGF gene in brown adipose tissue is independent of thermogenic oxygen consumption

To examine whether cold-induced vascular endothelial growth factor (VEGF) gene expression in brown adipose tissue involved generation of hypoxic oxygen levels by thermogenic processes, we cold-exposed wild-type mice, as well as uncoupling protein-1 (UCP1)-ablated mice in which no thermogenesis in brown adipocytes can be induced. Cold exposure stimulated VEGF expression in both wild-type and UCP1-ablated mice. Unexpectedly, the effect was 3-fold higher in UCP1-ablated animals, whereas cultured brown adipocytes from both genotypes responded identically to norepinephrine stimulation. These results demonstrate that generation of low oxygen levels does not contribute to cold-induced VEGF expression in brown adipose tissue, but the results are consistent with an adrenergic regulation of expression.

Norepinephrine but not hypoxia stimulates HIF-1α gene expression in brown adipocytes

The cellular response to hypoxic stress is mainly mediated via activation of the transcription factor hypoxia-inducible factor-1α (HIF-1α). In the present study, the sympathetically controlled brown adipose tissue was used to investigate the effect of norepinephrine on HIF-1α gene expression. Norepinephrine increased HIF-1α mRNA levels in cultured brown adipocytes, whereas the hypoxia-mimic cobalt was without effect. Cold exposure of mice increased HIF-1α gene expression in brown adipose tissue. In UCP1-ablated mice, which are incapable of inducing thermogenic oxygen consumption in brown adipose tissue, cold exposure generated a significantly higher elevation of HIF-1α mRNA levels than in wild-type. These results demonstrate that cold-induced HIF-1α gene expression is independent of thermogenic oxygen consumption leading to hypoxia, but is consistent with a norepinephrine regulation of HIF-1α gene expression. Thus, by elevating HIF-1α gene expression, norepinephrine may mediate an increased potential to respond to hypoxia in brown adipose tissue and possibly in other tissues.

Native UCP1 Displays Simple Competitive Kinetics between the Regulators Purine Nucleotides and Fatty Acids

Elucidation of the regulation of uncoupling protein 1 (UCP1) activity in its native environment, i.e. the inner membrane of brown-fat mitochondria, has been hampered by the presence of UCP1-independent, quantitatively unresolved effects of investigated regulators on the brown-fat mitochondria themselves. Here we have utilized the availability of UCP1-ablated mice to dissect UCP1-dependent and UCP1-independent effects of regulators. Using a complex-I-linked substrate (pyruvate), we found that UCP1 can mediate a 4-fold increase in thermogenesis when stimulated with the classical positive regulator fatty acids (oleate). After demonstrating that the fatty acids act in their free form, we found that UCP1 increased fatty acid sensitivity approximately 30-fold (as compared with the 1.5-fold increase reported earlier based on nominal fatty acid values). By identifying the UCP1-mediated fraction of the response, we could conclude that the interaction between purine nucleotides (GDP) and fatty acids (oleate) unexpectedly displayed simple competitive kinetics. In GDP-inhibited mitochondria, oleate apparently acted as an activator. However, only a model in which UCP1 is inherently active (i.e."activating" fatty acids cannot be included in the model), where GDP functions as an inhibitor with a K(m) of 0.05 mm, and where oleate functions as a competitive antagonist for the GDP effect (with a K(i) of 5 nm) can fit all of the experimental data. We conclude that, when examined in its native environment, UCP1 functions as a proton (equivalent) carrier in the absence of exogenous or endogenous fatty acids.

Uncoupling protein-1: involvement in a novel pathway for beta-adrenergic, cAMP-mediated intestinal relaxation.

The pathway for adrenergic relaxation of smooth muscle is not fully understood. As mitochondrial uncoupling protein-1 (UCP1) expression has been reported in cells within the longitudinal smooth muscle layer of organs exhibiting peristalsis, we examined whether the absence of UCP1 affects adrenergic responsiveness. Intestinal (ileal) segments were obtained from UCP1-ablated mice and from wild-type mice (C57Bl/6, 129/SvPas, and outbred NMRI). In UCP1-containing mice, isoprenaline totally inhibited contractions induced by electrical field stimulation, but in intestine from UCP1-ablated mice, a significant residual contraction remained even at a high isoprenaline concentration; the segments were threefold less sensitive to isoprenaline. Also, when contraction was induced by carbachol, there was a residual isoprenaline-insensitive contraction. Similar results were obtained with the beta(3)-selective agonist CL-316,243 and with the adenylyl cyclase stimulator forskolin. Thus the UCP1 reported to be expressed in the longitudinal muscle layer of the mouse intestine is apparently functional, and UCP1, presumably through uncoupling, may be involved in a novel pathway leading from increased cAMP levels to relaxation in organs exhibiting peristalsis.

Life without UCP1: mitochondrial, cellular and organismal characteristics of the UCP1-ablated mice.

Mice devoid of the original uncoupling protein UCP1 have provided opportunities to delineate UCP1 function in a series of biochemical and physiological contexts. The isolated brown-fat mitochondria from such mice are fully coupled (without the addition of GDP), but still exhibit a depressed capacity for ATP synthesis. However, they only show a 2-fold decrease in sensitivity to the de-energizing effect of free fatty acids, compared with UCP1-containing mitochondria, whereas they possess a (UCP1-independent) 50-fold higher sensitivity than liver mitochondria; the fatty acid sensitivities in wild-type and UCP1-deficient mitochondria may, however, be of different natures. Despite the fact that brown-fat cells from UCP1-ablated mice cannot produce heat when stimulated by noradrenaline ('norepinephrine') or fatty acids, UCP1-ablated mice can be induced to tolerate extended cold exposure, but the heat then fully results from shivering thermogenesis. Recruitable or adaptive (by cold acclimation or adaptation to a cafeteria diet) adrenergically-stimulated thermogenesis does not exist in the UCP1-ablated animals, demonstrating the unique ability of UCP1 to mediate recruitable non-shivering thermogenesis. In addition to information on the function of UCP1, the UCP1-ablated mice can be used to gain information concerning the function of the UCP1 homologues. Thus whereas an uncoupling function of the UCP1 homologues cannot be excluded, UCP1-ablated animals clearly lack any ability to recruit any UCP1 homologue to functionally replace the loss of thermogenesis resulting from UCP1. UCP1 (thermogenin) thus remains the only protein the activity of which can be recruited for the purpose of facultative thermogenesis.

Life without UCPI: mitochondrial, cellular and organismal characteristics of the UCPI-ablated mice

Mice devoid of the original uncoupling protein UCP1 have provided opportunities to delineate UCP1 function in a series of biochemical and physiological contexts. The isolated brown-fat mitochondria from such mice are fully coupled (without the addition of GDP), but still exhibit a depressed capacity for ATP synthesis. However, they only show a 2-fold decrease in sensitivity to the de-energizing effect of free fatty acids, compared with UCP1-containing mitochondria, whereas they possess a (UCP1-independent) 50-fold higher sensitivity than liver mitochondria; the fatty acid sensitivities in wild-type and UCP1-deficient mitochondria may, however, be of different natures. Despite the fact that brown-fat cells from UCP1-ablated mice cannot produce heat when stimulated by noradrenaline (‘norepinephrine’) or fatty acids, UCP1-ablated mice can be induced to tolerate extended cold exposure, but the heat then fully results from shivering thermogenesis. Recruitable or adaptive (by cold acclimation or adaptation to a cafeteria diet) adrenergically-stimulated thermogenesis does not exist in the UCP1-ablated animals, demonstrating the unique ability of UCP1 to mediate recruitable non-shivering thermogenesis. In addition to information on the function of UCP1, the UCP1 -ablated mice can be used to gain information concerning the function of the UCP1 homologues. Thus whereas an uncoupling function of the UCP1 homologues cannot be excluded, UCP1-ablated animals clearly lack any ability to recruit any UCP1 homologue to functionally replace the loss of thermogenesis resulting from UCP1. UCP1 (thermogenin) thus remains the only protein the activity of which can be recruited for the purpose of facultative thermogenesis.

Only UCP1 can mediate adaptive nonshivering thermogenesis in the cold.

Adaptive nonshivering thermogenesis may have profound effects on energy balance and is therefore therefore is a potential mechanism for counteracting the development of obesity. The molecular basis for adaptive nonshivering thermogenesis has remained a challenge that sparked acute interest with the identification of proteins (UCP2, UCP3, etc.) with high-sequence similarity to the original uncoupling protein-1 (UCP1), which is localized only in brown adipose tissue. Using UCP1-ablated mice, we examined whether any adaptive nonshivering thermogenesis could be recruited by acclimation to cold. Remarkably, by successive acclimation, the UCP1-ablated mice could be made to subsist for several weeks at 4C during which they had to constantly produce heat at four times their resting levels. Despite these extreme requirements for adaptive nonshivering thermogenesis, however, no substitution of shivering by any adaptive nonshivering thermogenic process occurred. Thus, although the existence of, for example, muscular mechanisms for adaptive nonshivering thermogenesis has recurrently been implied, we did not find any indication of such thermogenesis. Not even during prolonged and enhanced demand for extra heat production was any endogenous hormone or neurotransmitter able to recruit any UCP1-independent adaptive nonshivering thermogenic process in muscle or in any other organ, and no proteins other than UCP1-not even UCP2 or UCP3-therefore have the ability to mediate adaptive nonshivering thermogenesis in the cold.

Barbara Cannon's data on the UCP1-ablated mice: "non-cannonical" point of view.

The data of Cannon and co-workers on UCP1-ablated mice are interpreted assuming that UCP2 and UCP3 are involved in thermoregulation as fatty acid-dependent uncouplers although they are not sufficient, in the absence of UCP1, for long term maintenance of normal body temperature of mice after sudden and strong decrease in the ambient temperature. I would like to suggest that in brown fat of control mice, UCP1 is present in an amount higher than UCP2 and 3 and, therefore, is able to cause (a) some fatty acid-mediated decrease in proton motive force in resting state and, hence, (b) oxidation of CoQH2 to CoQ which is shown by Klingenberg and coworkers to be cofactor for UCPs. This results in strong uncoupling and thermogenesis mediated by UCP1, 2 and 3. In the UCP1-ablated mice, activity of UCP2 and 3 appears to be insufficient to induce CoQH2 oxidation in resting brown fat mitochondria, which results in hypothermia.