response to heat stress and other stresses in HepG2 cells and explored the underlying mechanisms. Results AMPK is inhibited by phosphatase 2A (PP2A) upon heat stress We first examined the effect of heat stress on AMPK activity in HepG2 cells by Western blot with antibodies that recognize phosphorylated Thr172 in AMPKa subunit (AMPKa). As shown in Figure 1A, exposure of HepG2 cells to 42uC induced rapid dephosphorylation of AMPKa, and the phosphorylated AMPKa, was almost undetectable at 1 h after heat stress. AMPKa phosphorylation gradually recovered after the cells were switched to 37uC. These results were consistent with the results from Hela and 293 cells [9]. We also examined the effect of heat stress on AMPKa phosphorylation in other cell types, including Hepa1-6, bEnd.3, C2C12, 293T and MIN6. Heat stress resulted in dephosphorylation of AMPKa in all these cell types (Figure 1B), suggesting that AMPK inhibition is a general event in cells under heat stress. As acetyl-CoA carboxylase (ACC) is a substrate for AMPK and phosphoenolpyruvate carboxykinase (PEPCK) gene expression is suppressed by AMPK activation, the phosphorylation of ACC and AMPK Regulates HSP70 mRNA level of PEPCK serve as indicators of AMPK activity. While exposure of HepG2 cells to 42uC for 30 min significantly induced ACC dephosphorylation (Figure 1C) and PEPCK mRNA expression (Figure 1D), pretreatment of the cells with AICAR (59 - aminoimidazole-4-carboxamide ribonucleoside), an AMPK spe- cific activator, reversed heat stress-induced dephosphorylation of ACC (Figure 1C) and upregulation of PEPCK expression (Figure 1D). These results suggest that the inhibition of AMPK activity under heat stress in turn causes ACC activation and PEPCK upregulation. As the activity of AMPK is modulated by intracellular AMP/ ATP ratio, we examined the ATP levels in HepG2 cells after exposure to 42uC for different time periods and found that heat stress for up to 4 h had no significant effect on intracellular level of ATP (Figure 1E and data not shown). This result suggests that the rapid dephosphorylation of AMPK under heat stress may result from other mechanism(s) than ATP change. It has been reported that PP2A could regulate the interaction between AMPKa2 and c1 subunits [11], and dephosphorylate AMPKa in cell-free assays [12]. Recently, PP2A was reported to mediate glucose-, palmitate-, or ethanol-induced AMPK inhibition [13–15]. To determine whether the inhibitory effect of heat stress on AMPK is mediated by activation of PP2A, we treated HepG2 cells with 100 nM okadaic acid (OA), a cell-permeable inhibitor of Figure 1. Heat stress induces AMPK dephosphorylation through activation of PP2A. A. HepG2 cells were exposed to 42uC (HS) for 0–1 h or 1 h followed by recovery at 37uC for 1–6 h. B. Various types of cells were exposed to 42uC (HS) for 30 min, then examined for AMPKa phosphorylation by Western blot. C–D. HepG2 cells pretreated with or without 1 mM AICAR for 15 min were exposed to 42uC (HS) for 30 min (C) or 1 h (D), then examined for ACC phosphorylation by Western blot (C), or PEPCK expression by real-time PCR (D). mean 6 SEM, n = 3. **P,0.01 vs. cells without heat stress; #P,0.05 vs. cells cultured in control medium under heat stress. E. HepG2 cells were exposed to 42uC (HS) for indicated times and measured intracellular ATP levels. mean 6 SEM, n = 3. F. HepG2 cells pretreated with 100 nM okadaic acid (OA) for 30 min or 1 mM AICAR for 15 min were exposed to 42uC (HS) for 30 min, then examined for AMPKa phosphorylation by Western blot. G–H. bEend.3 cells transfected with PP2Ac specific siRNA or control siRNA for 48 h were exposed to 42uC (HS) for 30 min. PP2Ac protein level (G) and AMPKa phosphorylation (H) were examined by Western blot. A–C and F–H: experiments were performed at least three times and the representative results are shown. doi:10.1371/journal.pone.0013096.g001 PLoS ONE | www.plosone.org 2 October 2010 | Volume 5 | Issue 10 | e13096