Resveratrol-Induced AMP-Activated Protein Kinase Activation Is Cell-Type Dependent: Lessons from Basic Research for Clinical Application

Despite the promising effects of resveratrol, its efficacy in the clinic remains controversial. We were the first group to report that the SIRT1 activator resveratrol activates AMP-activated protein kinase (AMPK) (Diabetes 2005; 54: A383), and we think that the variability of this cascade may be responsible for the inconsistency of resveratrol’s effects. Our current studies suggest that the effect of SIRT1 activators such as resveratrol may not be solely through activation of SIRT1, but also through an integrated effect of SIRT1-liver kinase B1 (LKB1)-AMPK. In this context, resveratrol activates SIRT1 (1) by directly binding to SIRT1; and (2) by increasing NAD+ levels by upregulating the salvage pathway through Nampt activation, an effect mediated by AMPK. The first mechanism promotes deacetylation of a limited number of SIRT1 substrate proteins (e.g., PGC-1). The second mechanism (which may be more important than the first) activates other sirtuins in addition to SIRT1, which affects a broad spectrum of substrates. Despite these findings, detailed mechanisms of how resveratrol activates AMPK have not been reported. Here, we show that (1) resveratrol-induced activation of AMPK requires the presence of functional LKB1; (2) Resveratrol increases LKB1 activity, which involves translocation and phosphorylation at T336 and S428; (3) Activation of LKB1 causes proteasomal degradation of LKB1; (4) At high concentrations (50–100 µM), resveratrol also activates AMPK through increasing AMP levels; and (5) The above-mentioned activation mechanisms vary among cell types, and in some cell types, resveratrol fails to activate AMPK. These results suggest that resveratrol-induced activation of AMPK is not a ubiquitous phenomenon. In addition, AMPK-mediated increases in NAD+ in the second mechanism require several ATPs, which may not be available in many pathological conditions. These phenomena may explain why resveratrol is not always consistently beneficial in a clinical setting

Resveratrol Prevents Oxidative Stress-Induced Senescence and Proliferative Dysfunction by Activating the AMPK-FOXO3 Cascade in Cultured Primary Human Keratinocytes

<div><p>The aging process is perceived as resulting from a combination of intrinsic factors such as changes in intracellular signaling and extrinsic factors, most notably environmental stressors. In skin, the relationship between intrinsic changes and keratinocyte function is not clearly understood. Previously, we found that increasing the activity of AMP-activated protein kinase (AMPK) suppressed senescence in hydrogen peroxide (H₂O₂)-treated human primary keratinocytes, a model of oxidative stress-induced cellular aging. Using this model in the present study, we observed that resveratrol, an agent that increases the activities of both AMPK and sirtuins, ameliorated two age-associated phenotypes: cellular senescence and proliferative dysfunction. In addition, we found that treatment of keratinocytes with Ex527, a specific inhibitor of sirtuin 1 (SIRT1), attenuated the ability of resveratrol to suppress senescence. In keeping with the latter observation, we noted that compared to non-senescent keratinocytes, senescent cells lacked SIRT1. In addition to these effects on H₂O₂-induced senescence, resveratrol also prevented the H₂O₂-induced decrease in proliferation (as indicated by ³H-thymidine incorporation) in the presence of insulin. This effect was abrogated by inhibition of AMPK but not SIRT1. Compared to endothelium, we found that human keratinocytes expressed relatively high levels of Forkhead box O3 (FOXO3), a downstream target of both AMPK and SIRT1. Treatment of keratinocytes with resveratrol transactivated FOXO3 and increased the expression of its target genes including catalase. Resveratrol’s effects on both senescence and proliferation disappeared when FOXO3 was knocked down. Finally, we performed an exploratory study which showed that skin from humans over 50 years old had lower AMPK activity than skin from individuals under age 20. Collectively, these findings suggest that the effects of resveratrol on keratinocyte senescence and proliferation are regulated by the AMPK-FOXO3 pathway and in some situations, but not all, by SIRT1.</p></div

Knockdown of FOXO3 attenuates the suppression of H₂O₂-induced senescence by resveratrol.

<p>(a) Keratinocytes were cultured in 12-well plates and at 30% confluence, were infected with lentivirus expressing shRNA to non-targeting control (shNegative) and FOXO3 (shFOXO3). Three days later, cells were treated with H₂O₂ ± resveratrol, further cultured for an additional 64 hrs. and then fixed with paraformaldehyde for SA-Gal staining, an indicator of senescence. Infection of shFOXO3 lentivirus suppressed FOXO3 expression by 70% but not FOXO1 expression. (b,c) Resveratrol suppressed H₂O₂-induced SA-Gal positive staining in shNegative infected cells, but this effect was largely diminished in shFOXO3 infected cells. * indicates p<0.05 compared to control treatment (n = 6).</p

AMPK activation is decreased with aging in human skin.

<p>Discarded skin was obtained from patients undergoing a surgical procedure. (a) The samples were processed for Western blot to evaluate AMPK activation levels using p-T172 AMPK and total AMPK (arrow) antibodies. GAPDH was used as a loading control. (b) The ratios of p-T172 to total AMPK were significantly lower in the samples from individuals over 50 years old (n = 6) compared to samples from individuals less than 20 years old (n = 6).</p

Effects of AMPK knockdown, AMPK inhibitor Compound C, SIRT1 inhibitor Ex527, and SIRT1 knockdown on ³H -thymidine incorporation.

<p>(a) Keratinocytes were infected with lentivirus vector expressing non-targeting control shRNA (shNegative) or AMPK alpha1 (shAMPKa1). Cells were then treated with 20 µM H₂O₂, 1 nM insulin and resveratrol (Res) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115341#pone.0115341.g005" target="_blank">Fig. 5</a>. Knockdown of AMPK abrogated the effects of resveratrol (which are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115341#pone.0115341.g005" target="_blank">Fig. 5c</a>). (b) Non-infected cells were treated with H₂O₂ and resveratrol as in 6a, but incubated with 1 µg/ml Compound C (CC), an AMPK inhibitor, 30 min. prior to the addition of resveratrol. Compound C inhibited the effects of resveratrol on ³H -thymidine incorporation. (c) Keratinocytes were infected with lentivirus vector expressing non-targeting control shRNA (shNegative) or SIRT1 (shSIRT1) that reduced total SIRT1 by about 70%. Knocking down SIRT1 had no effect on resveratrol-induced changes in ³H -thymidine incorporation. (d) Non-infected cells were incubated with H₂O₂, insulin and resveratrol as in 6a, and 10 µM Ex527 was added 10 min. prior to the addition of resveratrol. Ex527 treatment did not alter ³H -thymidine incorporation suggesting that inhibition of SIRT1 did not modulate the effect of resveratrol. For (a) and (c), numbers inside parenthesis denote n.</p

AMPK activation increases FOXO3 protein expression.

<p>(a) Keratinocytes grown in a 6-well plate were treated with the indicated concentrations of resveratrol for 30 min. Cells were harvested and subjected to Western blot. 25–50 µM resveratrol induced a 3–fold increase in FOXO3 protein expression. * indicates p<0.05 compared to 0 µM resveratrol treatment. (b) Human keratinocytes and umbilical vein endothelial cells were cultured in 6-well plates until 80% confluence. The cells were then incubated with the indicated reagents for 30 min.-2 hrs. Keratinocytes and endothelial cells were harvested simultaneously for Western blotting. Keratinocyte blotting required only a 30-second exposure for FOXO3 and a 5-minute exposure for FOXO1. Endothelial cell blotting required a 5-minute exposure for FOXO3 and only a 30-second exposure for FOXO1. This suggests that FOXO3 is richer in keratinocytes than in endothelium. Various AMPK activators, in particular 2-deoxyglucose and resveratrol, increased FOXO3 and FOXO1 proteins in keratinocytes. This effect was less apparent in endothelium.</p

Knockdown of FOXO3 suppresses resveratrol’s effects on ³H -thymidine incorporation.

<p>Cells were infected with the same lentivirus targeting FOXO3 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115341#pone.0115341.g004" target="_blank">Fig. 4</a>. Compared to infection with shNegative, infection with shFOXO3 resulted in a 50% reduction in ³H-thymidine incorporation. While resveratrol increased ³H-thymidine incorporation in shNegative infected cells, the effects were diminished in shFOXO3 infected cells.</p

Resveratrol increases FOXO3 transcriptional activity and expression of FOXO3 target genes.

<p>(a) FOXO3 transcriptional activity was assessed using the Fork Head Responsive Element (FHRE) reporter gene assay. Cells were grown in 24-well plates and transfected with the reporter plasmid at 40–50% confluence. The assay was performed 48 hrs. later. On the day of the assay, the cells were treated with H₂O₂ ± resveratrol for 6 hrs. after which they were harvested and luciferase activity was measured. Resveratrol induced a twofold increase in FOXO3 transcriptional activity. (b) The cells were incubated with the indicated concentrations of resveratrol for 30 minutes and harvested 16 hrs. later. mRNA levels were quantified by real-time PCR. Among the known FOXO3 target genes, expression of catalase (CAT) and BCL2-like 11 (BCL2L11) were significantly increased with 25 µM and 50 µM resveratrol and cell-cycle checkpoint protein cyclin G2 (CCNG2) and proapoptotic protein PUMA (BBC3) were increased by 50 µM resveratrol. * indicates p<0.05 compared to 0 µM resveratrol treatment.</p