Frohnert B I, Hui T Y, Bernlohr D A
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, St. Paul, Minnesota 55108, USA.
J Biol Chem. 1999 Feb 12;274(7):3970-7. doi: 10.1074/jbc.274.7.3970.
Fatty acid transport protein (FATP), a plasma membrane protein implicated in controlling adipocyte transmembrane fatty acid flux, is up-regulated as a consequence of adipocyte differentiation and down-regulated by insulin. Based upon the sequence of the FATP gene upstream region (Hui, T. Y., Frohnert, B. I., Smith, A. J., Schaffer, J. A., and Bernlohr, D. A. (1998) J. Biol. Chem. 273, 27420-27429) a putative peroxisome proliferator-activated receptor response element (PPRE) is present from -458 to -474. To determine whether the FATP PPRE was functional, and responded to lipid activators, transient transfection of FATP-luciferase reporter constructs into CV-1 and 3T3-L1 cells was carried out. In CV-1 cells, FATP-luciferase activity was up-regulated 4- and 5.5-fold, respectively, by PPARalpha and PPARgamma in the presence of their respective activators in a PPRE-dependent mechanism. PPARdelta, however, was unable to mediate transcriptional activation under any condition. In 3T3-L1 cells, the PPRE conferred a small but significant increase in expression in preadipocytes, as well as a more robust up-regulation of FATP expression in adipocytes. Furthermore, the PPRE conferred the ability for luciferase expression to be up-regulated by activators of both PPARgamma and retinoid X receptor alpha (RXRalpha) in a synergistic manner. PPARalpha and PPARdelta activators did not up-regulate FATP expression in 3T3-L1 adipocytes, however, suggesting that these two subtypes do not play a significant role in differentiation-dependent activation in fat cells. Electromobility shift assays showed that all three PPAR subtypes were able to bind specifically to the PPRE as heterodimers with RXRalpha. Nuclear extracts from 3T3-L1 adipocytes also showed a specific gel-shift complex with the FATP PPRE. To correlate the expression of FATP to its physiological function, treatment of 3T3-L1 adipocytes with PPARgamma and RXRalpha activators resulted in an increased uptake of oleate. Moreover, linoleic acid, a physiological ligand, up-regulated FATP expression 2-fold in a PPRE-dependent manner. These results demonstrate that the FATP gene possesses a functional PPRE and is up-regulated by activators of PPARalpha and PPARgamma, thereby linking the activity of the protein to the expression of its gene. Moreover, these results have implications for the mechanism by which certain PPARgamma activators such as the antidiabetic thiazolidinedione drugs affect adipose lipid metabolism.
脂肪酸转运蛋白（FATP）是一种参与控制脂肪细胞跨膜脂肪酸通量的质膜蛋白，它会随着脂肪细胞分化而上调，并受胰岛素下调。根据FATP基因上游区域的序列（Hui, T. Y., Frohnert, B. I., Smith, A. J., Schaffer, J. A., and Bernlohr, D. A. (1998) J. Biol. Chem. 273, 27420 - 27429），在-458至-474处存在一个假定的过氧化物酶体增殖物激活受体反应元件（PPRE）。为了确定FATP的PPRE是否具有功能以及是否对脂质激活剂有反应，将FATP - 荧光素酶报告基因构建体瞬时转染到CV - 1和3T3 - L1细胞中。在CV - 1细胞中，在各自激活剂存在的情况下，PPARα和PPARγ分别以PPRE依赖的机制使FATP - 荧光素酶活性上调4倍和5.5倍。然而，PPARδ在任何条件下都无法介导转录激活。在3T3 - L1细胞中，PPRE在前脂肪细胞中使表达有小幅但显著的增加，并且在脂肪细胞中使FATP表达有更强的上调。此外，PPRE赋予荧光素酶表达能够被PPARγ和视黄酸X受体α（RXRα）的激活剂协同上调的能力。然而，PPARα和PPARδ激活剂在3T3 - L1脂肪细胞中并未上调FATP表达，这表明这两种亚型在脂肪细胞的分化依赖性激活中不起重要作用。电泳迁移率变动分析表明，所有三种PPAR亚型都能够作为与RXRα的异二聚体特异性结合到PPRE上。3T3 - L1脂肪细胞的核提取物也显示出与FATP的PPRE有特异性的凝胶迁移复合物。为了将FATP的表达与其生理功能相关联，用PPARγ和RXRα激活剂处理3T3 - L1脂肪细胞导致油酸摄取增加。此外，生理配体亚油酸以PPRE依赖的方式使FATP表达上调2倍。这些结果表明，FATP基因拥有一个功能性的PPRE，并被PPARα和PPARγ的激活剂上调，从而将该蛋白的活性与其基因的表达联系起来。此外，这些结果对某些PPARγ激活剂如抗糖尿病噻唑烷二酮类药物影响脂肪脂质代谢的机制具有启示意义。
