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- Title
Resveratrol Activates AMP-Activated Protein Kinase (AMPK) by Increasing LKB1 Activity and AMP/ATP Ratio.
- Authors
Fan Lan; Schultz, Vera; Cacicedo, Jose M.; Ido, Yasuo
- Abstract
Resveratrol is a potent SirT1 (human homolog of silent information regulator 2) activator and is categorized into polyphenol class distributing in many plants such as green tea and grape. In previous ADA meetings, we demonstrated that resveratrol activated AMPK in LKB1 and SirT1-dependent manner. This study was undertaken to further elucidate the mechanism of resveratrol-induced AMPK activation. LKB1 activity was measured in immunoprecipitated samples by an enzymatic assay using LKBtide. In vivo LKB1 activity was assessed by blotting GST-LKB1 fusion protein expressed in 293T cells with the antibody against phospho-Thr336, an auto-phosphorylation site of LKB1. LKB1 translocation was assessed under fluorescence microscope by measuring GFP-LKB1 localization. Resveratrol at concentration as low as 25µM in 1hr increased GST-LKB1 Thr336 phosphorylation, suggesting activation of LKB1. AMPK and ACC (acetyl-CoA Carboxylase) phosphorylation was not apparent at this concentration but became evident at higher than 50 µM. AMP/ATP ratio calculated from ADP and ATP levels were not changed by 10 or 20 µM but were increased 5-fold by 50 µM and 10-fold by 100 µM resveratrol. In contrast, mitochondria uncoupler dinitrophenol increased AMP/ATP ratios, and AMPK and ACC phosphorylation, but had no effects on LKB1 Thr336 phosphorylation, suggesting that activation of LKB1 does not depend on AMP. LKB1 activation was also observed in the experiments using endothelial cells, in which 1hr treatment of 100µM resveratrol increased LKB1 activity by 100%, and this was inhibited by co-incubation of 100µM splitomicin, a SirT1 inhibitor. In 293T cells, resveratrol induced deacetylation of LKB1 and an increase in LKB1 Ser428 phosphorylation. 15µM resveratrol overnight treatment increased phosphorylation of GST-LKB1 Thr336 and ACC. And this was associated with 2-fold increase in translocation of LKB1 from nucleus to cytosol. SirT1 overexpression caused similar increases in phosphorylation of GST-LKB1 Thr336, AMPK and ACC and translocation of LKB1, but had no effects on AMP/ATP ratios. These results suggest 1) resveratrol increases LKB1 activity in SirT1-dependent mechanisms and 2) high concentration (greater than 50 µM) of resveratrol causes a SirT1-independent increase in AMP/ATP ratio, which induces robust activation of AMPK together with LKB1 activation.
- Subjects
RESVERATROL; ADENOSINE monophosphate; PROTEIN kinases; POLYPHENOLS; PHOSPHORYLATION; DINITROPHENOL; CYTOSOL
- Publication
Diabetes, 2007, Vol 56, pA514
- ISSN
0012-1797
- Publication type
Article