Long S D, Pekala P H
Department of Biochemistry, School of Medicine, East Carolina University, Greenville, North Carolina 27858, USA.
J Biol Chem. 1996 Jan 12;271(2):1138-44. doi: 10.1074/jbc.271.2.1138.
We have previously described the ability of arachidonic acid (AA) to regulate GLUT4 gene expression (Tebbey, P.W., McGowan, K.M., Stephens, J.M., Buttke, T.M., and Pekala, P.H. (1994) J. Biol. Chem. 269, 639-644). Chronic exposure (48 h) of fully differentiated 3T3-L1 cells to AA resulted in an approximately 90% suppression of GLUT4 mRNA accumulation. This decrease was demonstrated to be due to a 50% decrease in GLUT4 gene transcription as well as a destabilization of the GLUT4 message (t1/2 decreased from 8.0 to 4.6 h). In the current study we have identified, at least in part, the mechanism by which AA exerts its effects on GLUT4 expression. Compatible with a cyclooxygenase mediated event, the AA-induced suppression of GLUT4 mRNA was abolished by pretreating the cells with the inhibitor, indomethacin. Consistent with this observation, exposure of the cells to 10 microM PGE2 mimicked the effect of AA, in contrast to products of the lipoxygenase pathway which were unable to suppress GLUT4 mRNA content. Quantification of the conversion of AA to PGE2 demonstrated a 50-fold increase in PGE2 released into the media within 7 h of AA addition. Cyclic AMP levels were also increased 50-fold with AA treatment consistent with PGE2 activation of adenylate cyclase. Various long chain fatty acids, including the nonmetabolizable analog of AA, eicosatetraenoic acid (ETYA), also decreased GLUT4 mRNA levels. The effect of ETYA, a potent inhibitor of both lipo- and cyclooxygenases and a potent activator of peroxisome proliferator activated receptors (PPARs), suggested the presence of a second pathway where non-metabolized fatty acid functioned to suppress GLUT4 mRNA levels. Further support for a PPAR-mediated mechanism was obtained by exposure of the cells to the classic PPAR activator, clofibrate, which resulted in a approximately 75% decrease in GLUT4 mRNA content. Nuclear extracts prepared from the adipocytes contained a protein complex that bound to the PPAR responsive element (PPRE) found in the promoter of the fatty acyl-CoA oxidase gene. When the adipocytes were treated with either AA or ETYA, binding to the PPRE was disrupted, consistent with an ability of these fatty acids to control gene expression by altering the occupation of a PPRE. However, a perfect PPRE has yet to be identified in the GLUT4 promoter, but this does not rule the possibility of a PPAR playing an indirect role in the AA-induced GLUT4 mRNA suppression.
我们之前已经描述了花生四烯酸（AA）调节葡萄糖转运蛋白4（GLUT4）基因表达的能力（特贝，P.W.，麦高恩，K.M.，斯蒂芬斯，J.M.，巴特克，T.M.，以及佩卡拉，P.H.（1994年）《生物化学杂志》269卷，639 - 644页）。将完全分化的3T3 - L1细胞长期（48小时）暴露于AA会导致GLUT4 mRNA积累量大约降低90%。这种降低被证明是由于GLUT4基因转录减少50%以及GLUT4信使核糖核酸（mRNA）的稳定性下降（半衰期从8.0小时降至4.6小时）。在当前的研究中，我们至少部分地确定了AA对GLUT4表达发挥作用的机制。与环氧化酶介导的事件相符，用抑制剂吲哚美辛预处理细胞可消除AA诱导的GLUT4 mRNA抑制作用。与此观察结果一致，细胞暴露于10微摩尔/升的前列腺素E2（PGE2）模拟了AA的作用，相比之下，脂氧合酶途径的产物无法抑制GLUT4 mRNA含量。对AA向PGE2转化的定量分析表明，添加AA后7小时内释放到培养基中的PGE2增加了50倍。用AA处理后，环磷酸腺苷（cAMP）水平也增加了50倍，这与PGE2激活腺苷酸环化酶一致。各种长链脂肪酸，包括AA的不可代谢类似物二十碳四烯酸（ETYA），也降低了GLUT4 mRNA水平。ETYA是脂氧合酶和环氧化酶的有效抑制剂以及过氧化物酶体增殖物激活受体（PPARs）的有效激活剂，其作用表明存在第二条途径，即未代谢的脂肪酸发挥作用来抑制GLUT4 mRNA水平。通过使细胞暴露于经典的PPAR激活剂氯贝丁酯获得了对PPAR介导机制的进一步支持，这导致GLUT4 mRNA含量大约降低75%。从脂肪细胞制备的核提取物含有一种蛋白质复合物，该复合物与在脂肪酰基辅酶A氧化酶基因启动子中发现的PPAR反应元件（PPRE）结合。当脂肪细胞用AA或ETYA处理时，与PPRE的结合被破坏，这与这些脂肪酸通过改变PPRE的占据来控制基因表达的能力一致。然而，在GLUT4启动子中尚未鉴定出完美的PPRE，但这并不排除PPAR在AA诱导的GLUT4 mRNA抑制中发挥间接作用的可能性。
