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
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
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DOI：
10.1016/j.prostaglandins.2010.12.005
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发表时间：
2011-04-01
影响因子：
2.9
通讯作者：
Yokota, Kazushige
中科院分区：
文献类型：
--
作者：
Hossain, Mohammad Salim;Chowdhury, Abu Asad;Yokota, Kazushige
过氧化物酶体增殖激活受体(PPAR) γ是众所周知的脂肪细胞分化和成熟的主要调节因子。前列腺素（PG） D-2可在脂肪细胞中产生并脱水为J(2)系列PG，包括15-deoxy-Delta(12.14)-PGJ(2) （15d-PGJ(2)）和Delta(12)-PGJ(2)，它们通过激活PPAR γ发挥促脂肪prostanoids的作用。然而，在脂肪细胞的生命阶段，Delta(12)-PGJ(2)的定量测定尚未尝试。在这项研究中，我们开发了一种酶联免疫吸附试验，使用小鼠抗血清特异性Delta(12)-PGJ(2)。根据标准曲线，δ (12)-PGJ(2)的量可在0.5 pg至14.4 ng范围内测定。我们的抗血清不能识别大多数其他前列腺素，包括15d-PGJ(2)，而仅与不稳定的PGJ发生28%的交叉反应(2)。该免疫实验用于测定培养的3T3-L1脂肪细胞成熟阶段内源性δ (12)-PGJ(2)的形成。培养脂肪细胞形成内源性δ (12)-PGJ(2)的能力在成熟阶段早期逐渐增加，并且比15d-PGJ水平更高(2)。用阿司匹林或吲哚美辛（一种通用的环加氧酶抑制剂）处理培养细胞，如预期的那样显著减少成熟培养基中内源性δ (12)-PGJ(2)的产生。此外，我们单独评估了不同剂量的PGJ(2)系列在成熟阶段对脂肪形成的外源影响。虽然Delta(12)-PGJ(2)的效力略低于15d-PGJ(2)，但这些PGJ(2)系列中的每一个都有效地挽救了脂肪的积累和典型脂肪细胞标记物的基因表达，这些标记物在阿司匹林的存在下被减弱。综上所述，我们的研究结果表明，内源性δ (12)-PGJ(2)通过在培养脂肪细胞成熟阶段激活PPAR γ和15d-PGJ(2)，在很大程度上促进了脂肪生成程序的上调。(C) 2011爱思唯尔公司版权所有。
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.