Abstract
Arachidonic acid (AA, a proinflammatory fatty acid) in combination with iron promotes excess reactive oxygen species (ROS) production and exerts a deleterious effect on mitochondria. We have shown previously that activation of AMP-activated protein kinase (AMPK) protects hepatocytes from AA + iron-induced apoptosis. Resveratrol, a polyphenol in grapes, has beneficial effects mediated through SIRT1, LKB1, and AMPK. This study investigated the potential of resveratrol to protect against the mitochondrial impairment induced by AA + iron and the underlying mechanism for this cytoprotection. Resveratrol treatment inhibited apoptosis, ROS production, and glutathione depletion elicited by AA + iron in HepG2 cells. In addition, resveratrol attenuated superoxide generation in mitochondria and inhibited mitochondrial dysfunction induced by AA + iron. Overall, AMPK activation by resveratrol contributed to cell survival, as supported by the reversal of its restoration of mitochondrial membrane potential by either overexpression of a dominant-negative mutant of AMPKα or compound C treatment. Resveratrol increased inhibitory phosphorylation of glycogen synthase kinase-3β (GSK3β) downstream of AMPK, which contributed to mitochondrial protection and cell survival. Likewise, small interfering RNA knockdown of LKB1, an upstream kinase of AMPK, reduced the ability of resveratrol to protect cells from mitochondrial dysfunction. Furthermore, this LKB1-dependent mitochondrial protection resulted from resveratrol's poly(ADP-ribose)polymerase activation, but not SIRT1 activation, as supported by the experiment using 3-aminobenzamide, a poly(ADP-ribose)polymerase inhibitor. Other polyphenols, such as apigenin, genistein, and daidzein, did not activate AMPK or protect mitochondria against AA + iron. Thus, resveratrol protects cells from AA + iron-induced ROS production and mitochondrial dysfunction through AMPK-mediated inhibitory phosphorylation of GSK3β downstream of poly(ADP-ribose)polymerase-LKB1 pathway.
- ROS, reactive oxygen species
- AA, arachidonic acid
- 3-AB, 3-aminobenzamide
- ACC, acetyl-CoA carboxylase
- AICAR, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside
- AMPK, AMP-activated protein kinase
- CaMKK, calcium/calmodulin-dependent kinase kinase
- DCFH-DA, 2′,7′-dichlorofluorescein diacetate
- GSK3β, glycogen synthase kinase 3β
- MMP, mitochondrial membrane potential
- MnTBAP, Mn(III) tetrakis 4-benzoic acid porphyrin
- MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide
- PAR, poly(ADP-ribose)polymer
- PARP, poly(ADP-ribose)polymerase
- PI, propidium iodide
- Rh123, rhodamine 123
- SOD, superoxide dismutase
- GSH, glutathione
- siRNA, small interfering RNA
- l-NAME, l-nitro-arginine-methyl-ester
- TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling
- DMEM, Dulbecco's modified Eagle's medium
- FBS, fetal bovine serum
- PBS, phosphate-buffered saline
- FACS, fluorescence-activated cell sorting
- DN, dominant negative
- KM, kinase mutant
- Ad, adenovirus
- SB216763, 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione
- STO-609, 7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid
- NOS, nitric-oxide synthase
- MMP, mitochrondrial permeability.
Footnotes
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This work was supported by the World Class University project funded by the Korean government (Ministry of Education, Science and Technology Development) [Grant R32-2008-000-10098-0].
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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ABBREVIATIONS:
- Received June 8, 2009.
- Accepted July 20, 2009.
- © 2009 The American Society for Pharmacology and Experimental Therapeutics
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