Adipose differentiation-related protein (ADRP) is highly expressed in macrophages and human atherosclerotic lesions. We demonstrated that Toll-like receptor (TLR) 4-mediated signals, which are involved in atherosclerosis formation, enhanced the expression of ADRP in macrophages. Lipopolysaccharide (LPS) enhanced the ADRP expression in RAW264.7 cells or peritoneal macrophages from wild-type mice, but not in macrophages from TLR4-deficient mice. Actinomycin D almost completely abolished the LPS effect, whereas cycloheximide decreased the expression at 12 h, indicating that the LPS-induced ADRP expression was stimulated at the transcriptional level and was also mediated by new protein synthesis. LPS enhanced the ADRP promoter activity, in part, by stimulating activator protein (AP)-1 binding to the Ets/AP-1 element. In addition, preceding the increase of the ADRP mRNA, LPS induced the expression of interleukin (IL)-6, IL-1α, and interferon-β mRNAs, all of which stimulated the ADRP expression. Antibodies against these cytokines or inhibitors of c-Jun NH2-terminal kinase and nuclear factor (NF)-κB suppressed the ADRP mRNA level. Thus TLR4 signals stimulate the ADRP expression both in direct and indirect manners. Pycnogenol (PYC), an extract of French maritime pine, suppressed the expression of ADRP and the above-mentioned cytokines. PYC suppressed the ADRP promoter activity and enhancer activity of AP-1 and NF-κB, whereas it did not affect the LPS-induced DNA binding of these factors. In conclusion, TLR4-mediated signals stimulate the ADRP expression in macrophages while PYC antagonizes this process. PYC, a widely used dietary supplement, might be useful for prevention of atherosclerosis.

Although it is generally considered that the fatty acid effect on ADRP expression is mediated by peroxisome proliferator-activated receptors (PPARs), we identified here an additional molecular mechanism using the NMuLi mouse liver nonparenchymal cell line, which expresses PPAR␥ and ␦ but not ␣. Oleic acid (OA) and specific ligands for PPAR␥ and -␦ stimulated ADRP expression as well as the Ϫ2,090-bp ADRP promoter activity which encompasses the PPAR response element (PPRE) adjacent to an Ets/activator protein (AP)-1 site. When the AP-1 site was mutated, OA failed to stimulate the activity despite the presence of the PPRE, whereas ligands for PPAR␥ and -␦ did stimulate it and so did a PPAR␣ ligand under the coexpression of PPAR␣. DNA binding of AP-1 was stimulated by OA but not by PPAR ligands. Because we previously demonstrated that Pycnogenol (PYC), a French maritime pine bark extract, suppressed ADRP expression in macrophages partly by suppression of AP-1 activity, we tested the effect of PYC on NMuLi cells. PYC reduced the OAinduced ADRP expression along with suppression of lipid droplet formation. However, PYC neither suppressed the OA-stimulated ADRP promoter activity nor DNA binding of AP-1 but, instead, reduced the ADRP mRNA half-life. All these results indicate that the effect of OA on ADRP expression requires AP-1 as well as PPRE, and PYC suppresses the ADRP expression in part by facilitating mRNA degradation. PYC, a widely used dietary supplement, could be beneficial for the prevention of excessive lipid accumulation such as hepatic steatosis. lipid droplet; hepatic steatosis; adipose differentiation-related protein; activator protein-1; peroxisome proliferator-activated receptor CYTOSOLIC LIPID DROPLETS are physiologically important because they have specific function in various cell types or tissues, for example, as an energy reservoir in adipocytes, sites of storage and biosynthesis of eicosanoids in leukocytes, and sites of production of pulmonary surfactants in pneumocytes (44). It has been demonstrated that excessive lipid accumulation, not only in an adipose tissue but also in various nonadipose tissues, is closely related to a variety of pathological conditions, including insulin resistance, type 2 diabetes mellitus, cardiovascular diseases, and fatty liver or nonalcoholic steatohepatitis (2, 59), all of which are emerging as important clinical ands socioeconomical worldwide problems. Mechanisms of intracellular lipid droplet formation have, therefore, increasingly attracted clinical and scientific interests.A variety of proteins are associated with intracellular lipid droplets (10). Among them, PAT family proteins, which comprise perilipin, adipose differentiation-related protein (ADRP), TIP47, S3-12 and OXPAT/MLDP, are implicated in the formation, stabilization, and metabolism of lipid droplets (8, 37). ADRP was first identified in the early stages of adipocyte differentiation (33). Later studies revealed that ADRP does not directly induce adipogenesis but instead facilitates uptake of fatty ac...