Until two decades ago, brown adipose tissue (BAT) was studied primarily as a thermogenic organ of small rodents in the context of cold adaptation. The discovery of functional human BAT has opened new opportunities to understand its physiological role in energy balance and therapeutic applications for metabolic disorders. Significantly, the role of BAT extends far beyond thermogenesis, including glucose and lipid homeostasis, by releasing mediators that communicate with other cells and organs. The field has made major advances by using new model systems, ranging from subcellular studies to clinical trials, which have also led to debates. In this perspective, we identify six fundamental issues that are currently controversial and comprise dichotomous models. Each side presents supporting evidence and, critically, the necessary methods and falsifiable experiments that would resolve the dispute. With this collaborative approach, the field will continue to productively advance the understanding of BAT physiology, appreciate the importance of thermogenic adipocytes as a central area of ongoing research, and realize the therapeutic potential.

Objectives: Several recent studies have indicated the presence of UCP1 in the kidney, challenging the paradigm that UCP1 is only found in brown and beige adipocytes and broadening the (patho)physiological significance of UCP1. The kidney localization has been the direct result of immunohistochemical investigations and an inferred outcome from multiple lines of reporter mice. These findings require confirmation and further physiological characterization. Methods: We examined UCP1 expression in the kidney using immunohistochemistry and qPCR. Transversal sections through or near the kidney hilum, consistently including perirenal brown fat and adjacent kidney tissue, were analyzed with four UCP1 antibodies. Results: In addition to detecting UCP1 in perirenal adipose tissue, we observed distinct immunopositive structures in the kidney with our in-house UCP1-antibody, ‘C10’, in apparent agreement with earlier reports. To corroborate this, we tested the C10-antibody on kidney sections from UCP1-ablated mice but found equal reactivity in these UCP1-negative tissues. We then tested the widely used antibody ab10983, previously employed in kidney studies. Also here, the positive signal persisted in UCP1-ablated mice, clearly invalidating earlier findings. UCP1 qPCR studies also failed to detect UCP1 mRNA above background. Finally, two highly specific antibodies, E9Z2V and EPR20381, accurately detected UCP1 in perirenal adipose tissue but showed no signal in the kidney. Conclusions: When appropriate controls are implemented, there is no evidence for the presence of UCP1 in the kidney. Consequently, this conclusion also implies that the results from UCP1 reporter mice, specifically regarding kidney expression of the UCP1 gene – though possibly applicable to other tissues – require reconfirmation before being accepted as evidence for the presence of UCP1 in non-adipose tissues.

The existence of the phenomenon of diet-induced thermogenesis—and its possible mediation by UCP1 in brown adipose tissue—has long been, and is presently, an important metabolic controversy. Particularly, several recent studies have failed to observe the hallmark of the phenomenon: augmentation of diet-induced obesity (i.e., fat mass) in UCP1-ablated mice, thus further casting doubt on the possible importance of this thermogenesis, for example in human metabolic control. However, scrutiny of the experimental details revealed important procedural differences between experiments that did not show or did show this augmentation of diet-induced obesity. Particularly, there were notable differences between the commercial diets used (Research Diets or Ssniff). We, therefore, tested to what degree these differences would suffice to explain the absence of a UCP1 effect. Wild-type mice fed Research Diets high-fat diet became obese, but UCP1-ablated mice became even more obese, as expected if UCP1-dependent diet-induced thermogenesis exists. Mice fed the Ssniff high-fat diet became less obese than those on the Research Diets food—and, importantly, no effect of UCP1 ablation was seen. The result with the Research Diets diet was fully due to differences in total fat mass and not explainable by differences in food intake. The two diets are different in carbohydrate (sucrose) and lipid (lard vs. palm oil) composition and in texture and taste. Probably some of these factors explain the difference, but the important conclusion is that when an appropriate diet was offered, the body weight manifestation of the phenomenon of UCP1-dependent diet-induced thermogenesis was a reproducible phenomenon, the existence of which may have significance also for human metabolic control.

In the cold, the absence of the mitochondrial uncoupling protein 1 (UCP1) results in hyper-recruitment of beige fat, but classical brown fat becomes atrophied. Here we examine possible mechanisms underlying this phenomenon. We confirm that in brown fat from UCP1-knockout (UCP1-KO) mice acclimated to the cold, the levels of mitochondrial respiratory chain proteins were diminished; however, in beige fat, the mitochondria seemed to be unaffected. The macrophages that accumulated massively not only in brown fat but also in beige fat of the UCP1-KO mice acclimated to cold did not express tyrosine hydroxylase, the norepinephrine transporter (NET) and monoamine oxidase-A (MAO-A). Consequently, they could not influence the tissues through the synthesis or degradation of norepinephrine. Unexpectedly, in the cold, both brown and beige adipocytes from UCP1-KO mice acquired an ability to express MAO-A. Adipose tissue norepinephrine was exclusively of sympathetic origin, and sympathetic innervation significantly increased in both tissues of UCP1-KO mice. Importantly, the magnitude of sympathetic innervation and the expression levels of genes induced by adrenergic stimulation were much higher in brown fat. Therefore, we conclude that no qualitative differences in innervation or macrophage character could explain the contrasting reactions of brown versus beige adipose tissues to UCP1-ablation. Instead, these contrasting responses may be explained by quantitative differences in sympathetic innervation: the beige adipose depot from the UCP1-KO mice responded to cold acclimation in a canonical manner and displayed enhanced recruitment, while the atrophy of brown fat lacking UCP1 may be seen as a consequence of supraphysiological adrenergic stimulation in this tissue.

An increase in mitochondrial DNA (mtDNA) mutations and an ensuing increase in mitochondrial reactive oxygen species (ROS) production have been suggested to be a cause of the aging process (the mitochondrial hypothesis of aging). In agreement with this, mtDNA-mutator mice accumulate a large amount of mtDNA mutations, giving rise to defective mitochondria and an accelerated aging phenotype. However, incongruously, the rates of ROS production in mtDNA mutator mitochondria have generally earlier been reported to be lower - not higher - than in wildtype, thus apparently invalidating the mitochondrial hypothesis of aging. We have here re-examined ROS production rates in mtDNA-mutator mice mitochondria. Using traditional conditions for measuring ROS (succinate in the absence of rotenone), we indeed found lower ROS in the mtDNA-mutator mitochondria compared to wildtype. This ROS mainly results from reverse electron flow driven by the membrane potential, but the membrane potential reached in the isolated mtDNA-mutator mitochondria was 33 mV lower than that in wildtype mitochondria, due to the feedback inhibition of succinate oxidation by oxaloacetate, and to a lower oxidative capacity in the mtDNA-mutator mice, explaining the lower ROS production. In contrast, in normal forward electron flow systems (pyruvate (or glutamate) + malate or palmitoyl-CoA + carnitine), mitochondrial ROS production was higher in the mtDNA-mutator mitochondria. Particularly, even during active oxidative phosphorylation (as would be ongoing physiologically), higher ROS rates were seen in the mtDNA-mutator mitochondria than in wildtype. Thus, when examined under physiological conditions, mitochondrial ROS production rates are indeed increased in mtDNA-mutator mitochondria. While this does not prove the validity of the mitochondrial hypothesis of aging, it may no longer be said to be negated in this respect. This paper is dedicated to the memory of Professor Vladimir P. Skulachev.

Perfluorooctanoic acid (PFOA) is a synthetic organofluoride surfactant associated with several toxic effects in humans and animals. Particularly, it has been observed that PFOA treatment of mice results in weight loss associated with recruited brown adipose tissue (BAT), including an increased amount of uncoupling protein 1 (UCP1). The molecular mechanism behind this BAT recruitment is presently unknown. To investigate the existence of possible cell-autonomous effects of PFOA, we treated primary cultures of brown and white (inguinal) adipocytes with PFOA, or with the non-fluorinated equivalent octanoate, or with vehicle, for 48 h (from day 5 to day 7 of differentiation). PFOA in itself increased the gene expression (mRNA levels) of UCP1 and carnitine palmitoyltransferase 1A (CPT1α) (thermogenesis-related genes) in both brown and white adipocytes. In addition, PFOA increased the expression of fatty acid binding protein 4 (FABP4) and peroxisome proliferator-activated receptor α (PPARα) (adipogenesis-related genes). Also the protein levels of UCP1 were increased in brown adipocytes exposed to PFOA. This increase was more due to an increase in the fraction of cells that expressed UCP1 than to an increase in UCP1 levels per cell. The PFOA-induced changes were even more pronounced under simultaneous adrenergic stimulation. Octanoate induced less pronounced effects on adipocytes than did PFOA. Thus, PFOA in itself increased the levels of thermogenic markers in brown and white adipocytes. This could enhance the energy metabolism of animals (and humans) exposed to the compound, resulting in a negative energy balance, leading to diminished fitness.

Cold acclimation and pharmacological peroxisome proliferator-activated receptor γ (PPARγ) activation have each earlier been shown to recruit brown adipose tissue (BAT) and beige adipocytes thermogenic machinery, enhancing uncoupling protein 1 (UCP1)-mediated thermogenic capacity. We here investigated whether cold acclimation and PPARγ agonism combined have additive effects in inducing brown and beige adipocytes UCP1 content and whether this translates into a higher thermogenic capacity and energy expenditure. C57BL/6J mice treated or not with pioglitazone (30 mg/kg/day) were maintained at 21°C or exposed to cold (7°C) for 15 days and evaluated for thermogenic capacity, energy expenditure and interscapular BAT (iBAT) and inguinal white adipose tissue (iWAT) mass, morphology, UCP1 content and gene expression, glucose uptake and oxygen consumption. Cold acclimation and PPARγ agonism combined synergistically increased iBAT and iWAT total UCP1 content and mRNA levels of the thermogenesis-related proteins PGC1a, CIDEA, FABP4, GYK, PPARa, LPL, GLUTs (GLUT1 in iBAT and GLUT4 in iWAT), and ATG when compared to cold and pioglitazone individually. This translated into a stronger increase in body temperature in response to the β₃-adrenergic agonist CL316,243 and iBAT and iWAT respiration induced by succinate and pyruvate in comparison to that seen in either cold-acclimated or pioglitazone-treated mice. However, basal energy expenditure, BAT glucose uptake and glucose tolerance were not increased above that seen in cold-acclimated untreated mice. In conclusion, cold acclimation and PPARγ agonism combined induced a robust increase in brown and beige adipocytes UCP1 content and thermogenic capacity, much higher than each treatment individually. However, our findings enforce the concept that increases in total UCP1 do not innately lead to higher energy expenditure.

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 minutes) 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 minutes) 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.

The potential ability of nutritional compounds to induce or enhance the browning of adipocytes has attracted large interest as a workable means of combatting the obesity epidemic. Green tea compounds are discussed as such inducers of an enhanced thermogenic capacity and activity. However, the cell-autonomous effects of green tea compounds on adipocytes have until now only been demonstrated in adipogenic cell lines (3T3-L1 and 3T3-F442A), i.e., cells of undefined tissue lineage. In this study, we examine the ability of green tea compounds to cell-autonomously induce thermogenic recruitment in authentic brown and brite/beige adipocytes in vitro. In primary brown adipocytes, the green tea compounds suppressed basal UCP1 gene expression, and there was no positive interaction between the compounds and adrenergic stimulation. In white adipocytes, green tea compounds decreased both basal and norepinephrine-induced UCP1 mRNA levels, and this was associated with the suppression of cell differentiation, indicated by reduced lipogenic gene expression and lipid accumulation. A lack of interaction between rosiglitazone and green tea compounds suggests that the green tea compounds do not directly interact with the PPARγ pathway. We conclude that there is a negative effect of the green tea compounds on basal UCP1 gene expression, in both brown and white primary adipocytes, in contrast to the positive effects earlier reported from studies in adipogenic cell lines. We posit that the epigenetic status of the adipogenic cell lines is fundamentally different from that of genuine brown and white adipocytes, reflected, e.g., in several-thousand-fold differences in UCP1 gene expression levels. Thus, results obtained with adipogenic cell lines cannot unreservedly be extrapolated as being relevant for authentic effects in brown and white adipocytes. We suggest that this conclusion can be of general concern for studies attempting to establish physiologically relevant cell-autonomous effects.

New Findings What is the topic of this review? It has been suggested that human brown adipose tissue (BAT) is more similar to the brite/beige adipose tissue of mice than to classical BAT of mice. The basis of this is discussed in relationship to the physiological conditions of standard experimental mice.