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 AMPKαβγ 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 AMPKα and AMPKβ subunits. In experiments using C-terminal truncation mutants of AMPKα (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 AMPKα 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 AMPKα 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.

Highlights

  • The ␤ and ␥ subunits have regulatory function

  • Consistent with results obtained from experiments that utilized cells cultured with H2O2 (Fig. 2), the increase in kinase activity produced by direct incubation of the AMPK␣␤␥ complex with H2O2 was associated with oxidative modification of the ␣ and ␤ subunits, as shown by a decrease in BIAM adduct formation as well as increased S-glutathionylation of the AMPK␣ and AMPK␤ subunits (Fig. 4, C and D)

  • These results indicate that H2O2 can directly increase AMPK activity and that such activation is accompanied by oxidative modification of cysteine residues within the AMPK␣ and AMPK␤ subunits

Read more

Summary

EXPERIMENTAL PROCEDURES

Mice—Male C57BL/6, C3HeB/FeJ, or acatalasemic C3Ga.Cg-Cat B/J mice, 8 –12 weeks of age, were purchased from Jackson Laboratory (Bar Harbor, ME). Cell extracts or lung extracts (0.4 mg/sample) obtained from control, acatalasemic, or ATZ-treated mice were incubated with BIAM (200 ␮M) for 30 min at room temperature, and excess BIAM was removed by passing the extracts through BioGel P10. BIAM-protein adducts were extracted from streptavidin agarose by boiling in Laemmli sample buffer for 10 min and subjected to reducing SDSPAGE and Western blot analysis with antibodies to the AMPK␣, AMPK␤, or AMPK␥ subunits. Expressed FLAG-AMPK␣, WT, or truncation mutants were purified using anti-M2 FLAG-agarose beads as described previously [54] and subjected to Western blot analysis with anti-FLAGM2 or anti-phospho-Thr172-AMPK antibodies. Beads containing FLAGAMPK␣ (e.g. WT or truncation mutants) were washed twice with kinase buffer, and AMPK activity was determined using a radiometric assay and SAMS peptide as a substrate.

We next determined whether
RESULTS
AMPK Activity Is Increased in the Lungs of Acatalasemic
Findings
DISCUSSION
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call