Endogenous synthesis of prostacyclin was positively regulated during the maturation phase of cultured adipocytes

Prostacyclin alternatively called prostaglandin (PG) I-2 is an unstable metabolite synthesized by the arachidonate cyclooxygenase pathway. Earlier studies have suggested that prostacyclin analogues can act as a potent effector of adipose differentiation. However, biosynthesis of PGI(2) has not been determined comprehensively at different life stages of adipocytes. PGI(2) is rapidly hydrolyzed to the stable product, 6-keto-PGF(1 alpha), in biological fluids. Therefore, the generation of PGI(2) can be quantified as the amount of 6-keto-PGF(1 alpha). In this study, we attempted to develop a solid-phase enzyme-linked immunosorbent assay (ELISA) using a mouse antiserum specific for 6-keto-PGF(1 alpha). According to the typical calibration curve of our ELISA, 6-keto-PGF(1 alpha) can be quantified from 0.8 pg to 7.7 ng in an assay. The evaluation of our ELISA revealed the higher specificity of our antiserum without the cross-reaction with other related prostanoids while it exhibited only the cross-reaction of 1.5 % with PGF(2 alpha). The resulting ELISA was applied to the quantification of 6-keto-PGF(1 alpha) generated endogenously by cultured 3T3-L1 cells at different stages. The cultured cells showed the highest capability to generate 6-keto-PGF(1 alpha) during the maturation phase of 4-6 days, which was consistent with the coordinated changes in the gene expression of PGI synthase and the IP receptor for PGI(2). Following these events, the accumulation of fats was continuously promoted up to 14 days. Thus, our immunological assay specific for 6-keto-PGF(1 alpha) is useful for monitoring the endogenous levels of the unstable parent PGI(2) at different life stages of adipogenesis and for further studies on the potential association with the up-regulation of adipogenesis in cultured adipocytes.