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. 2012 May 2;287(28):23489–23501. doi: 10.1074/jbc.M112.365874

FIGURE 3.

FIGURE 3.

SIRT1 activation by PKA is AMPK-dependent. A, activity of human recombinant purified SIRT1 (0.2 unit) was measured using a fluorometric assay after performing a kinase assay with the catalytic subunit of PKA in the presence or absence of 200 μm ATP. B, AMPK activation was measured by immunoblot using anti-Thr(P)-172 antibody in different cell lines after treatment with 10 μm forskolin (FSK) for different times. Compound C (CC; 10 μm) and (Rp)-cAMP (Rp; 100 μm) were added 2 h prior to the addition of forskolin. C, SIRT1 activity in A549 cells was measured after a 2-h incubation with A769662 (100 μm), AICAR (2 mm), and oligomycin (5 μm). Activity was expressed as the percentage of activity with respect to the control. *, p < 0.05 (ANOVA test, n = 3). AMPK activation by the different compounds was confirmed by Western blot (right) with anti-Thr(P)-172 antibody. D, determination of intracellular NAD+ levels in A549 cells treated as described in C. E, SIRT1 activity was measured in MEFs from WT, AMPK KO, and SIRT1 KO mice. Cells were incubated with A769662 (100 μm), AICAR (2 mm), or oligomycin (5 μm) for 2 h before measuring SIRT1 activity. SIRT1 activity was normalized to the respective control for each cell type. SIRT1 KO cells showed no detectable activity. * and **, p < 0.05 (ANOVA test, n = 3). F, SIRT1 activity was determined in MEFs from WT mice. The AMPK inhibitor compound C (10 μm) was added to the cells 2 h before starting the treatments. Cells were incubated with A769662 (100 μm), A769662 + compound C, AICAR (2 mm), AICAR + compound C, oligomycin (5 μm), and oligomycin + compound C for 2 h before measuring SIRT1 activity. SIRT1 activity was normalized to control. * and **, p < 0.05 (ANOVA test, n = 3). G, intracellular NAD+ levels in WT MEFs treated as described in F. H, AMPK was inhibited in HepG2 cells by a pretreatment with compound C (10 μm) for 2 h, and SIRT1 activity was assessed after stimulation with 10 μm forskolin for 10 min. Activity is shown as -fold change with respect to the control. *, p < 0.05 (ANOVA test, n = 3). I, SIRT1 activity was measured in AMPK WT and KO (α1α2) MEFS treated with 10 μm forskolin for the indicated times. *, p < 0.05 (ANOVA test, n = 3). Error bars represent S.D. AFU, arbitrary fluorescence units; P-AMPK, phosphorylated AMPK.