Development of enzyme-linked immunosorbent assay for Δ 12-prostaglandin J 2 and its application to the measurement of the endogenous product generated by cultured adipocytes during the maturation phase

Abstract

Peroxisome proliferator-activated receptor (PPAR)γ 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-Δ 12,14-PGJ 2 (15d-PGJ 2) and Δ 12-PGJ 2, which serve as pro-adipogenic prostanoids through the activation of PPARγ. However, the quantitative determination of Δ 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 Δ 12-PGJ 2. According to the standard curve, the amount of Δ 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 Δ 12-PGJ 2 in cultured 3T3-L1 adipocytes during the maturation phase. The ability of cultured adipocytes to form endogenous Δ 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 Δ 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 Δ 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 Δ 12-PGJ 2 contributes substantially to the up-regulation of adipogenesis program through the activation of PPARγ together with 15d-PGJ 2 during the maturation phase of cultured adipocytes.