the presence of consensus sex steroid responsive elements within the UCP1 sequence is very intriguing. Indeed, there are several reports considering sex steroid hormones as modulator of the thermogenic capacity and activity of BAT (Monjo et al., 2003; Valle et al., 2008). It follows that the different effects of the UCP1 haplotypes could be explained by considering changes in the regulation of metabolism with aging, possibly mediated by age-related variations in hormonal status. Moreover, as suggested by Fessele et al. (2002), the importance of the sequence context in determining the functionality of a regulatory region clearly emerges. In fact, although the two poly- morphisms did not alter either the binding site for progesterone or estradiol, a clear haplotype-specific effect on the transcriptional activity was observed following hormonal stimulation.

222018161210Fold Induction4035302520Fold Induction= SS aFold Inductionxiv2.5@ basal conditionsRetinoic acid 1M (24h)  90.027p=0.03,p= 0.01670.046p=0.01 I| — apGL3 G-C G-A A-CReporter constructsBasal conditionsEstradiol 1M (48 h)p<o.00p<0.001 p-0.028;pros= —_pGL3 G-C G-A A-CReporter constructsBasal conditionsProgesterone 30nM (36h)p-0014p=0.021P0049p=0.026pGL3 G-C G-A A-C

-3826G allele have a diminished resting energy expenditure, while carriers of the G/G genotype have reduced thermoregulatory sympathetic nervous system (SNS) activity. This finding suggests that the attenuation of UCP1-mediated thermogenesis may have a negative effect on energy balance regulation. The survival advantage exerted by the A-C haplotype could then be a consequence of protection towards age-related decline of energy expenditure. These results seem to be in line with those reported by Rizzo et al. (2005) showing that very old individuals have a higher metabolic rate than older individuals. Human cohort studies also support a relationship between advanced age and body temperature (Waalen and Buxbaum, 2011), suggesting that the individual's ability to maintain a lower steady state body temperature may affect longevity. Although skeletal muscles may play a role in non shivering thermogenesis (Wijers et al., 2008), the age-related diminished thermogenic activity of BAT might be the primary cause for the loss of thermoregulation (Son'Kin et al., 2010). It is evident that an analysis of the BMI would be crucial to better understand how the effect of UCP1 activity on longevity is exerted. Unfortunately, information on BMI was available only for a subgroup of our sample. Therefore, we cannot fully address the question whether or not the effect of UCP1 on longevity is mediated through BMI or not. Further studies are needed to clarify this point.

On the other hand, since the preservation of mitochondrial function from oxidative stress represents one of the most important factors for achieving survival for the very old, the specific involvement of UCP1 in reducing ROS production (Oelkrug et al., 2010; Dlasková et al., 2010) may represent a further mechanism through which mitochondrial uncoupling in BAT can contribute to slow down aging. The hypothesis that UCP1 haplotypes affect ROS production is currently object of our investigation.

The present results, together with previous reports on UCP1, -2 and -3 (Crocco et al., 2011; Mookerjee et al., 2010) suggest that the uncoupling process is very important to conferring metabolic efficiency and, consequently, successful aging.

5. Conclusions

In conclusion, our study shows that the haplotypic variants in the 5′-enhancer region of the UCP1 gene modulate survival, probably by affecting the protein levels. This is the first study reporting a significant association between UCP1 genetic variants and human survival, however additional data are needed. On the other hand, the soundness of our preliminary findings is supported by the consistency between our association and in vitro studies and previous studies in model organisms.

Fig. 4. Luciferase activity of the reporter constructs at basal and after stimulation. A) MCF-7 cells treated with 1 μM of Retinoic Acid for 24 h; B) MCF-7 cells treated with 1 μM Estradiol for 48 h; C) T47D cells treated with 30 nM of Progesterone for 30 h. The values reported are the means±standard error mean of three independent experiments.

Acknowledgments

The work was supported by Fondi di Ateneo Unical (ex 60%) to GP and GR.

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