The procedures are described in the Supporting Materials. Western blotting, immunoprecipitation, reverse transcriptase-polymerase chain Selleckchem BAY 80-6946 reaction (RT-PCR), and measurement of ATP, NAD+/NADH ratio, and fatty acid (FA) uptake, are described in the Supporting Materials. First we tested whether RORα affected the activity of AMPK by measuring phosphorylation

at the threonine 172 residue of AMPK. Overexpression of RORα in HepG2 cells increased the amount of phosphorylated (p)AMPK, but not the expression level of AMPK. As ACC is inactivated by phosphorylation at serine 79 after AMPK activation, we measured the level of pACC. We found that the increase in the level of pAMPK was accompanied by the up-regulation of pACC. Treatment with CS, an activator of RORα, also induced the phosphorylation of AMPK and ACC, in a dose-dependent manner (Fig. 1A). Knockdown of RORα using siRNA did not increase either pAMPK or pACC in the presence of CS, indicating that CS activates AMPK via the activation of RORα (Fig. 1B). AMPK is activated by upstream kinases, such as the serine–threonine kinase liver MLN0128 in vitro kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase β. 6, 7 We observed that overexpression of RORα or CS treatment increased pLKB1 levels (Fig. 1C). However, treatment with STO609, an inhibitor of Ca2+ signaling, did not affect the RORα- or CS-induced activation of AMPK (Supporting Fig.

1), suggesting that RORα activates AMPK via the activation of LKB1, but not via Ca2+/calmodulin-dependent protein kinase kinase β. Infection of HepG2 cells with the adenovirus (Ad)-RORα virus or treatment with CS led to a significant decrease in ATP levels. The NAD+/NADH

ratio was consistently increased after Ad-RORα infection or CS treatment (Fig. 1D). These biochemical changes may cause the elevation of pLKB1 and the subsequent activation of AMPK after activation of RORα. Next we asked whether LXRα activity was altered in the presence of RORα. Treatment of HepG2 cells with TO901317 or GW3965, activating ligands of LXRα, led to induction of the expression level of LXRα and its target gene products, SREBP-1c and FAS. However, these increases were abolished by overexpression of RORα or CS treatment (Fig. 2A and Supporting Fig. 2A). Knockdown of RORα expression by siRORα abolished this CS-induced Miconazole repression, indicating that RORα mediates the effect of CS (Fig. 2B). Reporter gene assays employing LXR response element (LXRE)-tk-Luc and SRE-tk-Luc provided additional evidence that RORα decreases transcriptional activity of LXRα (Fig. 2C and Supporting Fig. 2B). Both overexpression of RORα in, and CS treatment of, HepG2 cells decreased the mRNA levels of LXRα, SREBP-1c, and FAS (Fig. 3A). A luciferase reporter encoding the 3-kb-long upstream promoter of LXRα was activated by TO901317; however, the induction was inhibited in the presence of RORα.