tion and/or the activity of other enzymes such as fatty acid synthase, sterol regulatory element-binding protein 1, and 3-hydroxy-3methylglutarl-coenzyme A Crenolanib web reductase. However, neither shizukaol D nor metformin could alter cellular palmitic acids content after 12 hours incubation. The exposure of HepG2 cells to high glucose for 24 h induces an 6 Shizukaol D Inhibits AMPK-Dependent Lipids Content doi: 10.1371/journal.pone.0073527.g004 insulin-resistant state and a decrease in both AMPK and ACC phosphorylation . In addition, our results agree with previously published studies showing that high glucose concentrations dramatically increase the triglyceride content in HepG2 cells but do not dramatically increase the cholesterol content . Furthermore, shizukaol D restored the levels of both AMPK and ACC phosphorylation that had been reduced by high glucose concentrations. Because treatment with shizukaol D inhibits the triglyceride and cholesterol content in HepG2 cells in the presence of either low glucose or high glucose, we propose that shizukaol D can lower the lipid content in HepG2 cells in both normal and insulin-resistant states. To confirm the significance of AMPK in the activity of shizukaol D, we inhibited AMPK using an AMPKa1 siRNA and the AMPK inhibitor compound C. AMPKa1 siRNA knocks down the expression of AMPKa1, an important subunit of AMPK that has a phosphorylation site on a conserved loop at Thr 172. A previous study showed that AMPKa1 knockdown inhibited the ability of metformin to activate AMPK and down-regulate lipid content. Compound C causes a remarkable inhibition of AMPK activity. Here, we observed that both AMPKa1 siRNA and compound C decreased the shizukaol D-mediated phosphorylation of AMPK and abrogated the ability of shizukaol D to reduce lipid levels. This 22988107 finding suggests that the modulation of lipid metabolism by shizukaol D is largely dependent on the AMPK-ACC signaling pathway. A number of AMPK activators, such as metformin, TZDs, and berberine, are known to generate mitochondrial dysfunction in cells. Here, we show that shizukaol D also decreased the mitochondrial membrane potential of HepG2 cells HepG2 cells were incubated with shizukaol D for 10 min, and the mitochondrial membrane potential was measured. Treatment with CCCP was used as a positive control. HepG2 cells were treated with shizukaol D at the indicated concentrations for 1 h, and then the AMP/ATP ratio was measured. The cells were treated with 2 M shizukaol D for the indicated time-points, and then the AMP/ATP ratio was measured. , p<0.05; , p<0.01 compared to the DMSO control. doi: 10.1371/journal.pone.0073527.g005 5A), although we did not detect the expression of any apoptotic markers in response to the drug treatment. AMPK activation is a direct result of alterations in the AMP/ATP ratio. Here, we found that treatment with shizukaol D increased the AMP/ATP ratio. Furthermore, shizukaol D inhibited cellular respiration, similar to metformin and rosiglitazone . We further investigated whether shizukaol D inhibits respiration in mitochondria isolated from 8825360 HepG2 cells . Surprisingly, we found that shizukaol D did not inhibit mitochondrial respiration using either complex I or complex II . This finding suggests that other factor may regulate aerobic respiration, such as the supply of electron donors . The inhibition of these factors may lead to the inhibition of aerobic respiration in cells, which would not be apparent in assays measuring the res