The exact molecular mechanism whereby C/EBPc regulates gene expression is not clear

aol 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 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 8832224 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 HepG2 cells . Surprisingly, we found that shizukaol D did not inhibit mitochondrial respiration using either MEK162 site 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 respiration of isolated mitochondria. Previous reports have shown that indomethacin, an antiinflammatory drug, suppresses glucose oxidation without affecting pyruvate oxidation in mitochondria. Furthermore, the compound C1 inhibits aerobic respiration but does not affect the activity of complex I or complex II in mitochondrial respiration. Our findings highlight the potential value of shizukaol D as a promising compound for the treatment of metabolic diseases by activating AMPK. 8 Shizukaol D Inhibits AMPK-Dependent Lipids 2173565 Content doi: 10.1371/journal.pone.0073527.g006 Induced pluripotent stem cells have been extensively studied in recent years since the groundbreaking discovery by a group from Kyoto University. The iPSCs were first reprogrammed from mouse somatic cells with the introduction of four transcription factors: Oct3/4, Sox2, Klf-4, and c-Myc . Since then, many groups have focused on finding the right formulation for making iPS cells that closely resemble embryonic stem cells and that satisfy all the standard definitions of pluripo