SIRTUIN EXPRESSION AS A FUNCTION OF TIME AND UVB IN EPIDERMAL KERATINOCYTES 61 Figure 1b, there is an abrogation of expression as a result of UVB irradiation indicating that, at a non-cytotoxic dose of UVB, as measured by the Alamar Blue assay for cell via- bility (data not shown), sirtuin expression is signifi cantly reduced. Thus, in the fi rst hours after irradiation, sirt 1, 3, and 6 are all reduced in contrast to their unirradiated controls. Sirt4 activity also changed after UVB exposure and instead of a reduced amount of activ- ity over the fi rst 4 hours, as seen in the controls, an overall increase that peaked at 8 h was also noted. Since SIRT4 inhibits the ability of glutam ate dehydrogenase to convert glu- tamate to α-ketoglutarate, leading to ATP synthesis, an increase in SIRT4 should con- tribute to reduced ATP levels. Inversely, impairment of SIRT3 activity by UVB exposure should also lead to reduced ATP levels due to a reduction in acetyl CoA synthetase activa- tion. Moreover, despite these changes, sirt6 recovered the fastest after 8 h again possibly due to its importance in DNA repair, genome maintenance, and infl ammation control. To determine the impact on energy production, the effects of 10 mJ/cm2 UVB on ATP and ROS were also measured in NHEK over time and correlated to sirtuin expression. Since sir- tuins are closely related to metabolism, we expected a strong effect on these after UVB expo- sure due to the loss of sirtuin expression. In Figure 2(A), a reduction in ATP was observed after UVB irradiation. Unexposed samples followed a temporal pattern similar to sirtuins 1, 3, and 6 expression and inversely to sirtuin 4 expression and demonstrate a correlation to metabolism. Alternatively, Figure 2(B) shows an increase in H2O2 in response to UVB com- pared to unexposed controls. Thus, after irradiation, an increase in H2O2 and a concomitant reduction in ATP were observed, which closely resembled the UVB-exposed expression pro- fi les for the sirtuins except for sirt6 at the 8 h time point. Signifi cant reduction in ATP as a result of UV exposure clearly correlates to changes in sirtuin transcription profi les. Figure 2. (A) ATP levels were plotted at 0, 2, 4, 6, 8, and 10 h after repletion with complete media and either exposed to UVB radiation or sham-irradiated. A temporal pattern similar to sirtuin expression was observed. However, UVB irradiation signifi cantly disrupted this pattern. (B) H2O2 levels in NHEK were plotted at 0, 2, 4, 6, 8, and 10 h after repletion with complete media and either exposed to UVB irradiation or sham-irradiated. UVB exposure signifi cantly increased H2O2 levels which were observed to modulate over time. Error bars were calculated for both graphs using S.E. measurement (n = 3).ABAB

JOURNAL OF COSMETIC SCIENCE 62 DISCUSSION Although most sirtuin research has focused on their effects on longevity, our approach has been to measure temporal changes in sirtuins in skin cells and also as a result of environ- mental challenge because the skin is the fi rst line of defense against the environment. Deleterious changes sustained over time in skin due to environmental insult can lead to photoag ing and increased visible signs of aging. We report here that sirt1 and sirt6 in NHEK follow a defi nite temporal pattern that is similar to the pattern that we had previ- ously observed for sirt3. These data support the fi ndings of Asher e t al. (11) who showed that SIRT1 follows a circadian pattern. Further, decreases in sirtuin expression after expo- sure to a non-cytotoxic dose of UVB were also measured which disrupted their temporal pattern. Since s irt6, unlike sirt1and 3, increased after 6 h under normal conditions and then was the fi rst to recover after UVB exposure, may indicate its importance for DNA repair (12). In parallel to these changes in sirtuin expression, we also observed a similar temporal pat- tern of ATP levels in NHEK. However, after irradiation, this pattern was signifi cantly altered, as in the sirtuins. Part of the reason for this may be attributable to a reduction in SIRT3 and the inactivation of acetyl CoA synthetase, which would normally lead to in- creased ATP synthesis (13). Additio nally, if SIRT3 is unable to deacetylate SOD2, there will be a concomitant increase in ROS (14) that we also observed. These results show how less obvious targets of sun exposure, such as sirtuins, may have a more subtle and long-term effect on skin. Our data present for the fi rst time a unifi ed modality for sirtuin expression and the effects that UVB has on their expression. Intrigu- ingly, the disruption of energy levels also points to the role that sirtuins play in metabo- lism and how low levels of UVB may quickly affect metabolic regulation. Taken together, these results further emphasize the importance of protection against sun exposure and may suggest a new paradigm for determining environmentally induced aging. Finally , our results show the importance of sustaining sirtuin levels and developing new cosmetic products in support of repair and metabolic mechanisms for a healthy skin. REFERENCES (1) Y. L. Deribe, T. Pawson, and I. Dikic, Post-translational modifi cations in signal integration, Nat. Struct. Mol. Biol., 17, 666–672 (2 010). (2) M. Kaeberlein, M. McVey, and L. Guarente, The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms, Genes Dev., 13, 2570–2580 ( 1999). (3) L. Guarente, Sir2 links chromatin silencing, metabolism, and aging, Genes Dev., 14, 1021–1026 (2000). (4) K. Dong, E. Pelle, D. B. Yarosh, and N. Pernodet, Sirtuin 4 identifi cation in normal human epidermal keratinocytes and its relation to sirtuin 3 and energy metabolism under normal conditions and UVB- induced stress, Exp. Dermatol., 21, 231–233 (2012). (5) J. T. McCarthy, E. Pelle, K. Dong, K. Brahmbhatt, D. Yarosh, and N. Pernodet, Effects of ozone in normal human epidermal keratinocytes, Exp. Dermatol., 22, 360–361 (2013). (6) A. A. Sauve, C. Wolberger, V. L. Schramm, and J. D. Boeke, The biochemistry of sirtuins, Annu. Rev. Biochem., 75, 435–465 (2006). (7) E. Pelle and N. Pernodet, “Sirtuin s: Biology and Anti-Aging Benefi ts for Skin Care,” in: Harry’s Cosme- tology, 9th Ed., M. Rosen. Ed. (in press). (8) T. G. Polefka, T. A. Meyer, P. P. Agin, and R. J. Bianchini, Effects of solar radiation on the skin, J. Cosmet. Dermatol., 11, 134–143 (2012). (9) M. C. Haigis and L. P. Guarente, Mammalian sirtuins–emerging roles in physiology, aging, and calorie restriction, Genes Dev., 20, 2913–2921 (2006).