Effects of WY-14,643 on the phosphorylation and activation of AMP-dependent protein kinase.
BACKGROUND
AMP-dependent protein kinase (AMPK) and peroxisome proliferator-activated receptor (PPAR) alpha facilitate fatty acid oxidation. We have shown that treatment of hepatoma cells with ethanol or feeding ethanol-containing diets to mice inhibited both PPARalpha and AMPK activity. Importantly, WY-14,643 reversed the development of fatty liver in alcohol-fed mice. Whether WY-14,643, a PPARalpha agonist, has any effects on AMPK is not known. The aim of this study was to investigate the effect of WY-14,643 on AMPK activity.


METHODS
The effect of WY-14,643 on AMPK phosphorylation and activity were examined in rat hepatoma cells (H4IIEC3). The effect of WY-14,643 on upstream kinases of AMPK, PKC-zeta/LKB1, intracellular AMP:ATP ratio, oxidative stress, and AMPK gene expression were studied.


RESULTS
Treatment of the H4IIEC3 cells with WY-14,643 for 24h led to 60% increase in the phosphorylation of AMPK. The effect of WY-14,643 on AMPK phosphorylation is PKC-zeta/LKB1 independent. WY-14,643 did not alter the levels of intracellular AMP:ATP ratio and it did not increase the levels of reactive oxygen species at 24-h of treatment. WY-14,643-induced AMPK alpha subunit expression by 2- to 2.5-fold, but there was no change in AMPKalpha subunit protein at 24h. The effect of WY-14,643 on AMPK phosphorylation did not altered by the presence of an NADPH oxidase inhibitor.


CONCLUSIONS
WY-14,643 induced AMPKalpha subunit phosphorylation and the activity of the enzyme. This was associated with induction of AMPKalpha1 and alpha2 mRNA, but the mechanism for this activation is uncertain.
Liangpunsakul
Suthat
S
Division of Gastroenterology and Hepatology, Department of Medicine, Indiana University School of Medicine and Roudebush Veterans Administration Medical Center, 545 N. Barnhill Drive, Indianapolis, IN 46202, USA.
Wou
Sung-Eun
SE
Wineinger
Kevin D
KD
Zeng
Yan
Y
Cyganek
Izabela
I
Jayaram
Hiremagalur N
HN
Crabb
David W
DW
D055372:AMP-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D002460:Cell Line; D004734:Energy Metabolism / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D047493:PPAR alpha / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism; D011743:Pyrimidines / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D000099221:Protein Kinase C zeta
D055372:AMP-Activated Protein Kinases; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D047493:PPAR alpha; D011493:Protein Kinase C; D011743:Pyrimidines; D017382:Reactive Oxygen Species; D000099221:Protein Kinase C zeta; C006253:pirinixic acid