Nilsson Rebecka, Ahmad Faiyaz, Swärd Karl, Andersson Ulrika, Weston Marie, Manganiello Vincent, Degerman Eva
Department of Experimental Medical Science, Lund University, BMC C11, 221 84 Lund, Sweden.
Cell Signal. 2006 Oct;18(10):1713-21. doi: 10.1016/j.cellsig.2006.01.010. Epub 2006 Feb 28.
Caveolae, plasma membrane invaginations particularly abundant in adipocytes, have been suggested to be important in organizing insulin signalling. Insulin-induced activation of the membrane bound cAMP degrading enzyme, phosphodiesterase 3B (PDE3B) is a key step in insulin-mediated inhibition of lipolysis and is also involved in the regulation of insulin-mediated glucose uptake and lipogenesis in adipocytes. The aim of this work was to evaluate whether PDE3B is associated with caveolae. Subcellular fractionation of primary rat and mouse adipocytes demonstrated the presence of PDE3B in endoplasmic reticulum and plasma membrane fractions. The plasma membrane PDE3B was further analyzed by detergent treatment at 4 degrees C, which did not solubilize PDE3B, indicating an association of PDE3B with lipid rafts. Detergent-treated plasma membranes were studied using Superose-6 chromatography which demonstrated co-elution of PDE3B with caveolae and lipid raft markers (caveolin-1, flotillin-1 and cholesterol) at a Mw of >4000 kDa. On sucrose density gradient centrifugation of sonicated plasma membranes, a method known to enrich caveolae, PDE3B co-migrated with the caveolae markers. Immunoprecipitation of caveolin-1 using anti caveolin-1 antibodies co-immunoprecipitated PDE3B and immunoprecipitation of flag-PDE3B from adipocytes infected with a flag-PDE3B adenovirus resulted in co-immunoprecipitation of caveolin-1. Studies on adipocytes with disrupted caveolae, using either caveolin-1 deficient mice or treatment of adipocytes with methyl-beta-cyclodextrin, reduced the membrane associated PDE3B activity. Furthermore, inhibition of PDE3 in primary rat adipocytes resulted in reduced insulin stimulated glucose transporter-4 translocation to caveolae, isolated by immunoprecipitation using caveolin-1 antibodies. Thus, PDE3B, a key enzyme in insulin signalling, appears to be associated with caveolae in adipocytes and this localization seems to be functionally important.
小窝是质膜内陷结构，在脂肪细胞中尤为丰富，有人认为它在组织胰岛素信号传导中起重要作用。胰岛素诱导膜结合的环磷酸腺苷（cAMP）降解酶磷酸二酯酶3B（PDE3B）活化，这是胰岛素介导的脂解抑制中的关键步骤，并且还参与脂肪细胞中胰岛素介导的葡萄糖摄取和脂肪生成的调节。这项工作的目的是评估PDE3B是否与小窝相关。对原代大鼠和小鼠脂肪细胞进行亚细胞分级分离，结果表明内质网和质膜级分中存在PDE3B。通过在4℃下用去污剂处理对质膜PDE3B进行进一步分析，去污剂未溶解PDE3B，表明PDE3B与脂筏相关。使用Superose-6色谱法研究经去污剂处理的质膜，结果表明PDE3B与小窝和脂筏标志物（小窝蛋白-1、 flotillin-1和胆固醇）在分子量>4000 kDa时共洗脱。在对超声处理的质膜进行蔗糖密度梯度离心（一种已知可富集小窝的方法）时，PDE3B与小窝标志物共同迁移。使用抗小窝蛋白-1抗体对小窝蛋白-1进行免疫沉淀可共免疫沉淀PDE3B，而从感染了flag-PDE3B腺病毒的脂肪细胞中对flag-PDE3B进行免疫沉淀则可共免疫沉淀小窝蛋白-1。使用小窝蛋白-1缺陷小鼠或用甲基-β-环糊精处理脂肪细胞对小窝破坏的脂肪细胞进行研究，降低了膜相关的PDE3B活性。此外，抑制原代大鼠脂肪细胞中的PDE3会导致胰岛素刺激的葡萄糖转运蛋白4向小窝的转位减少，小窝是使用小窝蛋白-1抗体通过免疫沉淀分离得到的。因此，胰岛素信号传导中的关键酶PDE3B似乎与脂肪细胞中的小窝相关，并且这种定位似乎在功能上很重要。
