Effects of WY-14,643 on the phosphorylation and activation of AMP-dependent protein kinase
Introduction
AMP-dependent protein kinase (AMPK)1 is a key enzyme in the regulation of energy metabolism and the response to cellular stress. Increases in intracellular AMP activate AMPK via several allosteric mechanisms [1]. The predominant effect is activation of LKB1 that activates AMPK by phosphorylating threonine 172 in the α subunit [1].
AMPK can also be activated by several AMP-independent pathways. Firstly, AMPK can be activated by Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) [2]. In some cell types, over-expression of CaMKKα increased AMPK activity, while pharmacological inhibition of CaMKKα nearly completely abolished AMPK activation [6]. Secondly, a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, transforming growth factor-activated kinase (TAK1) can also phosphorylate AMPK [3]. More recently, the ataxia telangiectasia mutated kinase (ATM kinase) was shown to phosphorylate AMPK in response to DNA damage [4].
Other physiological or pathological conditions that activate AMPK include oxidative stress [5]. Peroxynitrite caused phosphorylation of PKC-ζ in bovine aortic endothelial cells, with no effect on cellular AMP levels [6]. Choi et al. reported that H2O2 activated AMPK in NIH-3T3 cells by way of both increased AMP and a tyrosine kinase-dependent pathway [5]. These studies suggest that ROS might activate AMPK in some circumstances by activating other kinase cascades resulting in AMPK phosphorylation.
The activity of AMPK helps determine the fate of fatty acids. It reduces the activity of acetyl-CoA carboxylase (ACC). The net effect of AMPK activation is a reduction in the steady-state level of malonyl-CoA, which in turn increases the access of fatty acyl-CoA esters to the mitochondrial matrix space by removing the inhibition of carnitine palmitoyltransferase (CPT)-I [1]. Another factor regulating fatty acid metabolism is peroxisome proliferator-activated receptor alpha (PPARα). This nuclear factor is a major transcriptional regulator of fatty acid oxidation. Of note, PPARα actions are dependent on interactions with PPARγ coactivator 1α (PGC-1α) which was reported to be activated by phosphorylation by AMPK [7]. Synthetic PPARα agonists, WY-14,643 and clofibrate, have been used extensively to study PPARα-dependent cellular functions, but have been found to have a number of actions that may be independent of this receptor. For instance, in Kupffer cells, WY-14,643 activated NADPH oxidase and increased mRNA for TNFα[8]. WY-14,643 and clofibrate also increased the expression of CPT-1 and CPT-2 in Fao hepatoma cells and rat liver, respectively [9].
We have shown that treatment of hepatoma cells with ethanol or feeding ethanol-containing diets to mice inhibited both PPARα[10] and AMPK activity [11]. Furthermore, the amount of AMPKα subunit was decreased approximately 40% in the livers of ethanol-fed mice [11]. WY-14,643 reversed the development of fatty liver in alcohol-fed mice [10]. Since PPARα and AMPK seem to coordinate the regulation of lipid metabolism in the liver, the aim of this study was to investigate the effect of WY-14,643 on AMPK activity.
Section snippets
Materials
Most chemicals unless otherwise specified were purchased from Sigma Aldrich Chemical Company (St. Louis, MO) or Calbiochem-Novabiochem Corporation (San Diego, CA). Trypsin-EDTA, fetal bovine serum (FBS) and modified Eagle’s medium (MEM) were purchased from GIBCO Invitrogen Corporation, Inc (Carlsbad, CA). FBS charcoal-stripped of lipids was purchased from Hyclone Laboratories (Logan, UT). Radioisotopes were purchased from PerkinElmer Life and Analytical Sciences (Wellesley, MA). H4IIEC3
Activation of AMPK by WY-14,643 is independent of PKC-ζ/LKB1-phosphorylation
The effects of WY-14,643 on the phosphorylation and the activity of AMPK were examined in H4IIEC3 cells. The level of P-AMPK was normalized to the level of unphosphorylated AMPK to control for possible induction in enzyme mass. WY-14,643 significantly increased the P-AMPK/AMPK ratio, AMPK activity, and phosphorylation of its downstream target protein, ACC by 60%, 100%, and 15%, respectively, after 24 h treatment. Such an effect was not observed with 1–12 h treatment (Fig. 1). Several studies had
Discussion
This study provided evidence that AMPK can be phosphorylated and activated by the PPARα agonist WY-14,643. The mechanism did not involve changes in the phosphorylation of PKC-ζ/LKB1 and it did not depend on the generation of oxidative stress or changes in steady-state intracellular AMP:ATP ratio. We found that WY-14,643 increased expression of AMPK α1 and two subunit mRNAs, but we did not see a corresponding increase in total AMPK subunit (either AMPKα1 or α2) protein levels.
The role of LKB1 in
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