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Prostaglandin (PG) D(2) can be produced in adipocytes and dehydrated to PGs of J(2) series, including Δ(12)-PGJ(2) and 15-deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)), which serve as pro-adipogenic prostanoids through the activation of peroxisome proliferator-activated receptor γ. To accomplish the quantification of Δ(12)-PGJ(2) in the cell culture system of adipocytes, the present study aimed to develop a sensitive and specific immunological assay for Δ(12)-PGJ(2). Here, we established a cloned hybridoma cell line secreting a monoclonal antibody specifically recognizing Δ(12)-PGJ(2) and utilized for the development of its solid-phase enzyme-linked immunosorbent assay (ELISA). The immobilized antigen using a conjugate of Δ(12)-PGJ(2) and γ-globulin was competitively allowed to react with the monoclonal antibody in the presence of free Δ(12)-PGJ(2). The assay provided a sensitive calibration curve for Δ(12)-PGJ(2), allowing us to determine a range from 0.16 pg to 0.99 ng with a value of 13 pg at 50% displacement in one assay. The monoclonal antibody showed almost no cross-reactivity with other related prostanoids since PGJ(2) and 15d-PGJ(2) were only recognized with much lower values of 0.5% and 0.2%, respectively. The accuracy for determining Δ(12)-PGJ(2) in the culture medium of adipocytes was confirmed by measurement after the culture medium was fortified with known amounts of authentic Δ(12)-PGJ(2) in a range from 10 to 200 pg/mL. The application of our ELISA revealed that the formation of Δ(12)-PGJ(2) became more pronounced after several hours of incubation of PGD(2) at 37°C in fresh maturation medium of cultured adipocytes. Furthermore, we provide evidence for the increased ability of cultured adipocytes to synthesize endogenous Δ(12)-PGJ(2) during the progression of adipogenesis. These results indicate the reliability and usefulness of our solid-phase ELISA for stable Δ(12)-PGJ(2), reflecting the biosynthesis of unstable PGD(2) in the culture system of adipocytes.

15-deoxy-Δ¹²,¹⁴-prostaglandin J₂ (15d-PGJ₂) is a biologically active molecule serving as a pro-adipogenic factor or an anti-inflammatory regulator. This compound is one of naturally occurring derivatives formed by the non-enzymatic dehydration of PGD₂. To determine the endogenous synthesis of 15d-PGJ₂, a convenient immunological approach is useful. At first, we established a cloned hybridoma cell line to secrete a monoclonal antibody specific for 15d-PGJ₂. For the development of a solid-phase enzyme-linked immunosorbent assay (ELISA), the immobilized antigen using a protein conjugate of 15d-PGJ₂ was allowed to react competitively with a monoclonal antibody in the presence of free 15d-PGJ₂. Under the optimized conditions, a sensitive calibration curve was generated able to determine the amount of 15d-PGJ₂ from 0.5 pg to 9.7 ng with 71 pg of 50% displacement in one assay. Our monoclonal antibody did not recognize other related prostanoids except PGJ₂ with cross-reaction of 4%. Our ELISA was demonstrated to be reliable for the quantification of 15d-PGJ₂ in the maturation medium of cultured adipocytes by confirming the accuracy and specificity of its determination. The application of our assay revealed that the non-enzymatic formation of 15d-PGJ₂ became more evident after several hours of incubation with authentic PGD₂ at 37 °C. The results indicate the usefulness of our developed solid-phase ELISA with the monoclonal antibody for further studies on the endogenous synthesis of 15d-PGJ₂ and its roles in various cells and tissues.

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Abstract: Cultured preadipocytes enhance the synthesis of prostaglandin (PG) E2 and PGF2α involving the induction of cyclooxygenase (COX)-2 during the growth phase upon stimulation with a mixture of phorbol 12-myristate 13-acetate, a mitogenic factor, and calcium ionophore A23187. Here, we studied the interactive effect of 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) on the inducible synthesis of the endogenous PGs in cultured preadipocytes and its implication in adipogenesis program. 15d-PGJ2 interfered significantly the endogenous synthesis of those PGs in response to cell stimuli by suppressing the induction of COX-2 following the attenuation of NF-κB activation. In contrast, Δ12-PGJ2 and troglitazone had almost no inhibitory effects, indicating a mechanism independent of the activation of peroxisome proliferator-activated receptor γ for the action of 15-PGJ2. Pyrrolidinedithiocarbamate (PDTC), an NF-κB inhibitor, effectively inhibited on the inducible synthesis of those PGs in preadipocytes. Endogenous PGs generated by preadipocytes only during the growth phase in response to the cell stimuli autonomously attenuated the subsequent adipogenesis program leading to the differentiation and maturation of adipocytes. These effects were prevented by additional co-incubation of preadipocytes with either 15d-PGJ2 or PDTC although 15d-PGJ2 alone has no stimulatory effect. Moreover, 15d-PGJ2 did not block the inhibitory effects of exogenous PGE2 and PGF2α on the adipogenesis program in preadipocytes. Taken together, 15d-PGJ2 can interfere the COX pathway leading to the induced synthesis of endogenous PGs that contribute to negative regulation of adipogenesis program in preadipocytes. [Copyright &y& Elsevier]

Abstract: Adipocytes express preferentially lipocalin-type prostaglandin (PG)D synthase (L-PGDS) that is responsible for the biosynthesis of PGD2 and other related prostanoids with pro-adipogenic or anti-adipogenic effects. To evaluate the role of L-PGDS in cultured adipocytes and the precursor cells, we attempted to interfere the intracellular expression of L-PGDS in cultured 3T3-L1 preadipocytes by stable transfection with a mammalian expression vector having the full-length cDNA of L-PGDS oriented in the antisense direction. The cloned transfectants with antisense L-PGDS exhibited the reduction in the transcript and protein levels of L-PGDS, resulting in the significant inhibition of the PGD2 synthesis from exogenous and endogenous arachidonic acid. By contrast, the synthesis of PGE2 was not influenced appreciably, indicating no interfering effects on cyclooxygenases and PGE synthases. The stable transfection with antisense L-PGDS induced markedly the stimulation of fat storage in cultured adipocytes during the maturation phase. In addition, the spontaneous accumulation of fats occurred in the transfectants with antisense L-PGDS without undergoing the stimulation with inducing factors. The gene expression studies revealed the enhanced expression of adipocyte-specific markers in the transfectants with antisense L-PGDS, indicating the up-regulation of adipogenesis program. The stimulated adipogenesis was significantly reversed by anti-adipogenic prostanoids including PGE2 and PGF2α, while the storage of fats was additionally enhanced by pro-adipogenic 15-deoxy- Δ12,14-prostaglandin J2. These results suggest that the stably reduced expression levels of L-PGDS regulates positively adipogenesis program in a cellular mechanism independent of pro-adipogenic action of PGJ2 series. [Copyright &y& Elsevier]

Abstract: Peroxisome proliferator-activated receptor (PPAR)γ is a well-known master regulator for the differentiation and maturation of adipocytes. Prostaglandin (PG) D2 can be produced in adipocytes and dehydrated to J2 series of PGs including 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) and Δ12-PGJ2, which serve as pro-adipogenic prostanoids through the activation of PPARγ. However, the quantitative determination of Δ12-PGJ2 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 Δ12-PGJ2. According to the standard curve, the amount of Δ12-PGJ2 can be measured from 0.5pg to 14.4ng in an assay. Our antiserum did not recognize most other prostanoids including 15d-PGJ2, while it only showed the cross-reaction of 28% with unstable PGJ2. This immunological assay was applied to the determination of the endogenous formation of Δ12-PGJ2 in cultured 3T3-L1 adipocytes during the maturation phase. The ability of cultured adipocytes to form endogenous Δ12-PGJ2 increased gradually at an earlier stage of the maturation phase and detectable at higher levels than 15d-PGJ2. Treatment of cultured cells with either aspirin or indomethacin, a general cyclooxygenase inhibitor, significantly reduced the production of endogenous Δ12-PGJ2 in the maturation medium as expected. Furthermore, we evaluated individually the exogenous effects of PGJ2 series at various doses on adipogenesis during the maturation phase. Although Δ12-PGJ2 was slightly less potent than 15d-PGJ2, each of these PGJ2 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 Δ12-PGJ2 contributes substantially to the up-regulation of adipogenesis program through the activation of PPARγ together with 15d-PGJ2 during the maturation phase of cultured adipocytes. [Copyright &y& Elsevier]

15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) has been identified as a natural ligand for peroxisome proliferator-activated receptor (PPAR) γ to promote adipogenesis. However, it remains elusive about the ability of PPARγ-expressing adipocytes to produce PGJ2 series and the role in the life cycle of adipocytes. Here, we developed an enzyme-linked immunosorbent assay specific for 15d-PGJ2. The analysis using this method revealed the increase in the endogenous synthesis of immunoreactive 15d-PGJ2 in cultured adipocytes during the maturation phase. Further studies using cyclooxygenase inhibitors clarified the contribution of endogeous 15d-PGJ2 produced by mature adipocytes to upregulation of fat storage in an autocrine manner. [Copyright &y& Elsevier]