EXPRESSION OF TYPE III PHOSPHODIESTERASE IN NIH 3T3 CELLS

在分离的大鼠脂肪细胞中，激活一个cGMP抑制的环 核苷酸磷酸二酯酶（CGI PDE）是胰岛素的重要组成部分 这种激素的抗脂肪分解作用。 这种激活是 与CGI PDE上丝氨酸位点的磷酸化相关。 然而，这种激活的机制和功能意义 关于胰岛素的细胞作用还没有很好的理解。 最近，已克隆了大鼠脂肪细胞CGI PDE的cDNA（RcGIP 1 来自大鼠脂肪组织cDNA文库。 为了证明 该cDNA编码胰岛素敏感的CGI PDE，我们在 过度表达胰岛素受体的哺乳动物细胞。 的重组 将质粒RcGIP 1-pBPV（牛乳头瘤病毒启动子）转染到 3006细胞，一种稳定的NIH 3 T3成纤维细胞系，过表达 胰岛素受体 在筛选后获得的50个克隆中， 显示CAMP PDE活性被0.5 μ M抑制至少75% 西洛酰胺，CGI PDE家族的选择性抑制剂。 具体 这些阳性克隆的活性至少比对照高100倍。 在仅用pBPV转染的对照细胞中测量的比活性。 表达的PDE具有预测的分子量（约135-Kda） 并与亲和纯化的抗血小板CGI PDE抗体发生交叉反应。 用胰岛素处理转染细胞（10- 9 M，15分钟） 使粗匀浆的CAMP PDE活性增加30%至100%。 在微粒体组分中观察到胰岛素刺激265% (105 000 g沉淀）的一个阳性克隆中， 颗粒部分占原油总活性的80 匀浆。 这些结果证实了RcGIP 1 cDNA编码一种 胰岛素敏感膜相关CGI PDE。 工作正在进行中 为了确定观察到的胰岛素刺激是否涉及 酶的磷酸化。

In isolated rat adipocytes, activation of a CGMP-inhibited cyclic nucleotide phosphodiesterase (CGI PDE) by insulin is an important part of the antilipolytic action of this hormone. This activation is associated with phosphorylation of serine site(s) on the CGI PDE. However, the mechanism and the functional significance of this activation in terms of the cellular action of insulin is not yet well understood. Recently, the CDNA (RcGIP1) for the rat adipocyte CGI PDE has been cloned from rat adipose tissue CDNA libraries. In order to demonstrate that this CDNA encodes an insulin-sensitive CGI PDE we expressed this CDNA in mammalian cells overexpressing the insulin receptor. A recombinant plasmid RcGIP1-pBPV (bovine papilloma virus promoter) was transfected in 3006 cells, a stable cell line of NIH 3T3 fibroblasts overexpressing the insulin receptor. Among fifty clones obtained after screening, twelve exhibited CAMP PDE activity that is inhibited at least 75% by 0.5 microM cilostamide, a selective inhibitor of the CGI PDE family. Specific activities of these positive clones are at least 100-fold higher than the specific activity measured in control cells transfected by pBPV alone. The expressed PDE exhibited the predicted molecular mass (about 135-Kda) and cross-reacted with affinity purified anti-platelet CGI PDE antibody. Treatment of the transfected cells by insulin (10-9M for 15 min) increased the CAMP PDE activity of the crude homogenates by 30 to 100%. Stimulation of 265% by insulin was observed in the microsomal fraction (105,000 g pellet) of one positive clone where the activity of the particulate fraction represents 80% of the total activity of the crude homogenate. These results confirm that the RcGIP1 CDNA encodes for an insulin sensitive membrane-associated CGI PDE. Work is now in progress in order to determine whether the observed insulin stimulation involves phosphorylation of the enzyme.