**1. Introduction**

Uncoupling protein-3 (UCP3) is a mitochondrial protein, first discovered in 1997 [1], and prevalently expressed in skeletal muscle (SkM), the heart and brown and white adipose tissues [2]. The extent of homology between the UCP1 and UCP3 genes led to the proposal that UCP3 might be involved in thermogenic mechanisms, and although it does not appear to contribute to cold-induced thermogenesis [3], it has recently proved to be essential in thermogenic responses that are induced by the endotoxin lipopolysaccharide and by the sympathomimetic methamphetamine [4]. Indeed, the evidence that up-regulation of UCP3 is not always associated with mitochondrial uncoupling/thermogenesis [5], which suggested that uncoupling oxidative phosphorylation is not the primary role of UCP3, but rather a consequence of its true function. Other than thermogenesis, other roles that have been attributed to UCP3 include prevention of damage induced by reactive oxygen species (ROS) and lipid hydroperoxides (LOOH), as well as modulation of lipid handling [6–9]. Indeed, high amounts of UCP3 are present in tissues that are known to metabolize fatty acids (FA) to a high extent, and enhanced levels

of UCP3 expression are observed under physiological and pathological conditions, in which the fatty acid oxidation rate is elevated [8–10]. In addition, the expression of UCP3 during heart development is correlated to that of mitochondrial fatty acid oxidation rate markers [11]. Furthermore, the absence of UCP3 negatively influences the ability of SkM mitochondria to oxidize FA [12,13]. In this context, Bouillaud et al. [14] suggested that UCP3 could switch cells from carbohydrate to fatty acid metabolic pathways by promoting mitochondrial pyruvate extrusion, which prevents the use of pyruvate as a substrate. However, mechanistic information on this possible activity is currently lacking.

Although the roles that were proposed for UCP3 sugges<sup>t</sup> that it could play a role in energy homeostasis (EH), the obtained contrasting results have not produced a common and unambiguous conclusion so far. Several lines of experimental evidence supporting the role of UCP3 in EH came from human studies [see 6 and references within] and from mice over-expressing UCP3, being: (i) obesity-resistant mice present higher UCP3 levels than obesity-prone mice [15]; (ii) transgenic mice that over-express UCP3 are metabolically less e fficient than their wild-type litter mates, and are protected against high fat diet (HFD)-induced obesity [16,17]; and, (iii) modest UCP3 over-expression in SkM increased mice energy expenditure [18]. Conversely, some studies that were performed on mice lacking UCP3 (KO mice) are discordant, since they did not show alterations in several metabolic parameters, such as the resting metabolic rate, the regulation of body temperature during cold exposure, food intake, body weight regulation, and the total body triglyceride content [3,19]. Nevertheless, KO mice have been shown not to be obese [3] (only showing higher lipid accumulation relative to WT litter mates when fed a HFD for a prolonged period (eight months)). Furthermore, the KO mice showed no alteration in metabolic e fficiency [20]. The absence of metabolic alterations in KO mice could be due to the constant thermal stress that is caused by the animal housing conditions. In fact, in most studies, mice (which have a thermoneutrality temperature of 30 ◦C) are housed at the standard temperature (20–24 ◦C), which represents cold stress. Thus, mice lacking UCP3 that are exposed to thermal stress may implement compensatory mechanisms to maintain their body temperature, and these mechanisms are likely to a ffect the overall metabolic rate and other investigated parameters. Such a possibility was previously tested for UCP1 [21]. UCP1 ablation only induced obesity when the mice were housed under thermoneutral conditions. This outcome highlights the importance of avoiding thermal stress in metabolic studies on mice, since housing temperatures significantly influence the outcome of experiments, as well as their translatability to humans that, indeed, create a thermoneutral environment without cold stress for themselves [22]. Here, we investigated the role of UCP3 in metabolic control in situations in which thermal stress was eliminated. We report that, in adult mice acclimated at thermoneutrality for two weeks, UCP3 ablation altered energy and lipid metabolism. Moreover, in standard diet fed mice, which were kept at thermoneutrality since weaning, UCP3 ablation enhanced ectopic fat accumulation in liver and skeletal muscle, and vastly augmented HFD-induced fat accumulation. We conclude that the exposure temperature is determinative for the outcome of metabolic e ffects elicited by UCP3 and that the protein can be involved in the metabolic control of lipid metabolism in mice and possibly in humans.

#### **2. Materials and Methods**
