Development of enzyme-linked immunosorbent assay for Δ12-prostaglandin J2 and its application to the measurement of the endogenous product generated by cultured adipocytes during the maturation phase

Peroxisome proliferator-activated receptor (PPAR)gamma is a well-known master regulator for the differentiation and maturation of adipocytes. Prostaglandin (PG) D-2 can be produced in adipocytes and dehydrated to J(2) series of PGs including 15-deoxy-Delta(12.14)-PGJ(2) (15d-PGJ(2)) and Delta(12)-PGJ(2), which serve as pro-adipogenic prostanoids through the activation of PPAR gamma. However, the quantitative determination of Delta(12)-PGJ(2) has not been attempted during the life stage of adipocytes. In this study, we developed an enzyme-linked immunosorbent assay using mouse antiserum specific for Delta(12)-PGJ(2). According to the standard curve, the amount of Delta(12)-PGJ(2) can be measured from 0.5 pg to 14.4 ng in an assay. Our antiserum did not recognize most other prostanoids including 15d-PGJ(2), while it only showed the cross-reaction of 28% with unstable PGJ(2). This immunological assay was applied to the determination of the endogenous formation of Delta(12)-PGJ(2) in cultured 3T3-L1 adipocytes during the maturation phase. The ability of cultured adipocytes to form endogenous Delta(12)-PGJ(2) increased gradually at an earlier stage of the maturation phase and detectable at higher levels than 15d-PGJ(2). Treatment of cultured cells with either aspirin or indomethacin, a general cyclooxygenase inhibitor, significantly reduced the production of endogenous Delta(12)-PGJ(2) in the maturation medium as expected. Furthermore, we evaluated individually the exogenous effects of PGJ(2) series at various doses on adipogenesis during the maturation phase. Although Delta(12)-PGJ(2) was slightly less potent than 15d-PGJ(2), each of these PGJ(2) series rescued effectively both the accumulation of fats and the gene expression of typical adipocyte-markers that were attenuated in the presence of aspirin. Taken together, our findings indicate that endogenous Delta(12)-PGJ(2) contributes substantially to the up-regulation of adipogenesis program through the activation of PPAR gamma together with 15d-PGJ(2) during the maturation phase of cultured alipocytes. (C) 2011 Elsevier Inc. All rights reserved.