Exposure to Hydrogen Peroxide Induces Oxidation and Activation of AMP-activated Protein Kinase*

Abstract

Although metabolic conditions associated with an increased AMP/ATP ratio are primary factors in the activation of 5-adenosine monophosphate-activated protein kinase (AMPK), a number of recent studies have shown that increased intracellular levels of reactive oxygen species can stimulate AMPK activity, even without a decrease in cellular levels of ATP. We found that exposure of recombinant AMPK␣␤␥ complex or HEK 293 cells to H 2 O 2 was associated with increased kinase activity and also resulted in oxidative modification of AMPK, including S-glutathionylation of the AMPK␣ and AMPK␤ subunits. In experiments using C-terminal truncation mutants of AMPK␣ (amino acids 1-312), we found that mutation of cysteine 299 to alanine diminished the ability of H 2 O 2 to induce kinase activation, and mutation of cysteine 304 to alanine totally abrogated the enhancing effect of H 2 O 2 on kinase activity. Similar to the results obtained with H 2 O 2 -treated HEK 293 cells, activation and S-glutathionylation of the AMPK␣ subunit were present in the lungs of acatalasemic mice or mice treated with the catalase inhibitor aminotriazole, conditions in which intracellular steady state levels of H 2 O 2 are increased. These results demonstrate that physiologically relevant concentrations of H 2 O 2 can activate AMPK through oxidative modification of the AMPK␣ subunit. The present findings also imply that AMPK activation, in addition to being a response to alterations in intracellular metabolic pathways, is directly influenced by cellular redox status. AMPK3 is a serine/threonine kinase that consists of three subunits, of which the ␣ subunit has inducible kinase activity and the ␤ and ␥ subunits have regulatory function. Formation of the ␣␤␥ complex is required for optimal allosteric activation of AMPK, which is induced by binding of AMP to the ␥ subunit (1-4). In addition to activation by AMP, phosphorylation of the Thr 172 residue of the ␣ subunit enhances kinase activity (5, 6). Recent studies have shown that the autoinhibitory domain (AID), located between amino acids 312 and 335 of the AMPK␣ subunit, is responsible for the lack of kinase activity under basal conditions (7-9), whereas AMP-induced conformational changes within the ␣␤␥ complex diminish function of the AID and lead to kinase activation.The regulation of AMPK activity is primarily thought to result from alterations in the intracellular AMP/ATP ratio, arising from diminished ATP generation due to hypoxia, glucose deprivation, heat shock, or reduction in mitochondrial oxidative phosphorylation or from increased ATP consumption, such as occurs during strenuous exercise (2, 10 -12). Once activated, AMPK can phosphorylate and modulate the function of essential metabolic pathways participating in the regulation of glucose and lipid homeostasis (13-15). A major effect of AMPK activation is in preserving energy for use under conditions where ATP is limiting (4,16). AMPK activation appears to prevent or diminish inflammation-associated organ injury, including the development of a...

Activation of AMP-kinase by AICAR induces apoptosis of DU-145 prostate cancer cells through generation of reactive oxygen species and activation of c-Jun N-terminal kinase

“…AMPK activation through ROS has been previously demonstrated among others in endothelial cells (36), HepG2 hepatocarcinoma cells (37) and vascular smooth muscle cells (38) which may occur before or without alteration in the ATP/AMP ratio (38)(39)(40). In this respect it has been recently shown that physiologically relevant concentrations of H 2 O 2 can activate AMPK through oxidative modification of the AMPKα subunit, and it was discussed that AMPK activation, in addition to being a response to alterations in intracellular metabolic pathways, is directly influenced by cellular redox status (41). The data of the present study shed new light on the role of AMPK activity on cancer cell apoptosis.…”

Toll-Like Receptor 4 Engagement Inhibits Adenosine 5′-Monophosphate-Activated Protein Kinase Activation through a High Mobility Group Box 1 Protein-Dependent Mechanism

“…Treatment of mice with 5-aminoimidazole-4-carboxamide-1-β-Dribofuranoside (AICAR) or metformin, two inducers of AMPK activation, reduced the severity of lipopolysaccharide (LPS)-induced acute inflammatory lung injury (11,16). However, there is little evidence that AMPK is activated in inflammatory states, such as acute lung injury, despite the presence of conditions including increased release of reactive oxygen species and diminished generation of ATP, which would be expected to result in AMPK activation (9,(17)(18)(19). Therefore, a potentially important, and presently unanswered question, relates to the mechanisms that may prevent activation of AMPK in such conditions.…”

“…Classically, activation of AMPK has been described to occur under conditions of cellular stress that affect the balance between cellular adenosine 5′-triphosphate (ATP), adenosine 5′-diphosphate (ADP) and AMP and involves direct binding of AMP and ADP to the AMPK γ subunit, which then results in phosphorylation of Thr172 within the AMPK α activating loop (3)(4)(5)(6)(7)(8). Recent studies have shown that exposure of cells to reactive oxygen species or glycogen can induce AMPK activation independently of changes in cellular ATP-to-AMP ratios (9,10).…”

Activation of AMPK Enhances Neutrophil Chemotaxis and Bacterial Killing

“…Western Blot analysis was performed as described previously (44,45). Briefly, cell lysates of murine bone marrow neutrophils (3.5 × 10 6 /well) were prepared using lysis buffer containing Tris pH 7.4 (50 mmol/L), NaCl (150 mmol/L), NP-40 (0.5%, vol/vol), EDTA (1 mmol/L), EGTA (1 mmol/L), okadaic acid (1 nmol/L) and protease inhibitors.…”