6C-E). mRNA and protein levels learn more of Fsp27 and Cideb were not affected by the knockdown of SREBP1c (Fig. 6D and Supporting Fig. 8C-E). Importantly, mRNA and protein levels of Cidea were significantly reduced in the SREBP1c-knockdown ob/ob hepatocytes treated with PAs (Fig. 6C-E). Consistently, the hepatic TAG level was reduced in SREBP1c knock-down hepatocytes treated with PAs (Fig. 6F). In contrast, the knockdown of SREBP1c did not affect Cidea mRNA and protein levels (Fig. 6C-E) and hepatic TAG accumulation in the presence of OAs (Fig. 6F). These data indicate that SREBP1c is an important mediator of saturated FA-induced Cidea expression

and hepatic lipid accumulation. During the course of our analysis, we noted that the increase in the Cidea protein levels was higher than the corresponding increase in its mRNA levels in the presence of both saturated and unsaturated FAs (Fig. 5D), which suggested that Cidea protein stability may be increased in the presence of FAs. To test this possibility, we first treated primary ob/ob hepatocytes with OAs or PAs and then incubated them with cycloheximide (CHX), which

inhibits protein synthesis. OA treatment significantly prolonged the half-life of Cidea, which was increased from 40 to 80 minutes (Fig. 7A,B). Consistent c-Met inhibitor with our previous study using adipocytes,33 the half-life of Fsp27 in ob/ob hepatocytes was also increased in the presence of OAs (Fig. 7A,B). Half-lives of Cidea and Fsp27 in ob/ob hepatocytes were also increased in the presence of PAs (Supporting Fig. 9A,B). In contrast, Cideb was a relatively stable protein; its stability was not affected by FA treatment (Fig. 7A). Because FAs are usually converted into TAGs and stored in LDs, we checked whether FA-induced Cidea stability Phosphoglycerate kinase in hepatocytes was dependent on lipid synthesis

by knocking down diacylglycerol O-acyltransferase (DGAT)1 and DGAT2, which are enzymes that catalyze the final step of TAG synthesis. Levels of DGAT1/2 in ob/ob hepatocytes were decreased by small interfering RNAs (siRNAs) specific for DGAT1/2 (Fig. 7C), and levels of Cidea and Fsp27 proteins and their half-life also decreased significantly (Fig. 7D-F and Supporting Fig. 9C). Similar results were observed in AML12 cells that overexpressed HA-Cidea (Supporting Fig. 9D-F). These data indicated that Cidea and Fsp27 were stabilized by treatment with FFAs and by lipid synthesis, which provided a positive feedback mechanism that promoted lipid storage and liver steatosis in hepatocytes. CIDE family proteins are important regulators of various aspects of lipid metabolism, including control of lipid storage and LD size in adipocytes (by Cidea and Fsp27)15, 19 and control of very-low-density lipoprotein (VLDL) lipidation in the liver (by Cideb).