submitted a sequence of 463 bp (from −3967 to −3504) the two polymorphic sites to the MatInspector online software. shown in Fig. 3, a putative retinoic acid response element

accumulation, and thermogenesis in mice models but also to be associated with increased median survival and diminished age- related diseases (McDonald and Horwitz, 1999; Kontani et al., 2005; Gates et al., 2007).

the two polymorphic sites to the MatInspector online software. As shown in Fig. 3, a putative retinoic acid response element (from −3842 to −3826), including the A-3826G variation, and a previously characterized ATF/CREB binding element (from −3738 to −3733), carrying the C-3737A variation (Rousset et al., 2002), were predicted. Furthermore, a progesterone responsive element (PRE)-like sequence (from −3817 to −3804), and an estrogen responsive element (ERE)- like sequence (from −3713 to −3701), were also predicted in the vicinity of the two SNPs. Subsequently, using a luciferase reporter gene assay we generated reporter constructs carrying the three haplotypes and then tested their transcriptional activity in transiently transfected MCF-7 and T47D cells, which specifically express the estradiol and progesterone receptors, respectively. Experiments were carried out both under basal conditions and after hormonal stimulation. As a first step we estimated the maximum effective concentration by quantitative dose–response experiments (data not shown). Fig. 4 shows that, under basal conditions, in both cell lines, the luciferase activity of the cells carrying either the A-C or the G-A construct was significantly higher than cells carrying the haplotype G- C, which did not show any transcriptional activity. Moreover, the activity of the A-C construct was higher than that of G-A.

For these reasons, a number of nucleotide variants in the UCP1 gene have been described and analyzed in adult onset metabolic disorders (Mori et al., 2001; Herrmann et al., 2003; Kim et al., 2005, 2006). The A-3826G transition, which is in strong linkage disequilib- rium with the C-3737A polymorphism (Rousset et al., 2002), has been associated with the percentage of body fat gain over time, high weight gain during adult life, and increased BMI in obese subjects (see Jia et al., 2010 and references therein). Here we report a relationship between UCP1 variability and survival at very old age. In particular, we found that the frequency of the A-C/A-C diplotype increases, while the A-C/G-A frequency decreases as the population ages. By contrast, no variation in the A-C/G-C diplotype frequency was found. The A-3826G and the C-3737A polymorphisms are located at the 5′ region of the gene in proximity of a complex enhancer region (from position −3820 to −3470 upstream of the transcription start site) containing multiple and distinct cis-acting elements that mediate strong drug-dependent transcriptional activation of the UCP1 gene (del Mar Gonzales-Barroso et al., 2000). The results of our in vitro transfection experiments using luciferase reporter assays clearly point to an effect of these haplotypes on UCP1 gene expression. The A-C haplotype showed higher transcriptional activity compared to the G-A haplotype. No activity was observed for the GC haplotype. These results seem to indicate a major role for the A-3826G polymorphism in modulating the transcriptional activity of the UCP1 gene. In line with this, it has been shown that the -3826G allele is associated with reduced mRNA levels (Sramkova et al., 2007), while the C to A substitution at −3737, does not seem to substantially affect transcriptional activity (Rousset et al., 2002).

With respect to basal conditions, a higher luciferase activity was observed for both the A-C (p=0.027) and G-A (p=0.046) constructs in MCF-7 cells treated with 9-cis retinoic acid (1 μM) for 24 h. In contrast, A-C demonstrated a significantly decreased luciferase activity (p=0.028) when the same cells were treated with 17β- estradiol (1 μM) for 48 h (Fig. 4A and B). Moreover, in presence of 17β-estradiol the G-A construct also showed a trend for reduced activity which was not statistically inferior to basal transcription. In contrast, the activity of the sole A-C construct increased when T47D cells were treated with progesterone (30 nM) for 36 h (p=0.014; Fig. 4C).

It is worth noting that, although A-C and G-A haplotypes positively affect UCP1 transcriptional activity compared to the GC haplotype, they have opposite effects on longevity. This apparently contrasting result may be explained considering the haplotype-specific effect after hormonal stimuli. In fact, progesterone up-regulates whereas estradiol down-regulates the transcriptional activity of the AC haplotype, while neither changes the transcriptional activity of GA. Therefore, it is likely that the real in vivo effect of these haplotypes on complex phenotypes such as aging and longevity is mediated by the intricacy of the different cellular and physiological stimuli. Similarly, this may explain why the G-C haplotype, lacking any transcriptional activity, also did not show significant changes in its frequency during aging. It is evident that this observation calls for a better understand- ing of the different stimuli affecting UCP1 transcription. In this frame,

4. Discussion

Human aging is accompanied by changes in energy balance and body composition which are consequent to hormonal changes (Hughes et al., 2004). In this context, the role of BAT is particularly important, since it regulates energy expenditure in the elderly. In fact, although the amount of BAT in human adults is low, it is responsible for 1–2% of the energy expenditure, which is enough to prevent weight gain of about 2 kg per year (Lean, 1989). Consistently, an inverse relation between BAT and BMI, body fat, and visceral fat is observed in aging (Mattson, 2010; Vijgen et al., 2011). UCP1, which is expressed in BAT, has been observed to influence metabolism, fat