EXPRESSION OF TYPE III PHOSPHODIESTERASE IN NIH 3T3 CELLS

在分离的大鼠脂肪细胞中，CGMP抑制的环的激活 核苷酸磷酸二酯酶(CGI PDE)是胰岛素作用的重要组成部分 这种荷尔蒙的抗脂作用。此激活是 与CGI PDE上丝氨酸位点(S)的磷酸化相关。 然而，这种激活的机制和功能意义 就胰岛素的细胞作用而言，目前还不是很清楚。 最近，大鼠脂肪细胞CGI PDE的CDNA(RcGIP1)已被克隆 从大鼠脂肪组织CDNA文库中获得。为了证明 该CDNA编码胰岛素敏感的CGI PDE，我们在 哺乳动物细胞过度表达胰岛素受体。重组人 重组牛乳头瘤病毒启动子RcGIP1-pBPV NIH3T3成纤维细胞的稳定细胞系3006细胞过表达 胰岛素受体。在筛选后得到的50个克隆中，有12个 显示出0.5微米至少抑制75%的cAMP PDE活性 西洛胺，CGI PDE家族的选择性抑制剂。特定的 这些阳性克隆的活性至少是 在单独转染pBPV的对照细胞中测量比活性。 表达的PDE显示了预测的相对分子质量(约135-kda) 并与亲和纯化的抗血小板CGI PDE抗体发生交叉反应。 胰岛素对转基因细胞的作用(10~(-9)M，15分钟) 使粗匀浆中cAMP-PDE活性提高30%~100%。 在微粒体组分中观察到胰岛素刺激265%。 (105,000克颗粒)，其中一个阳性克隆的活性 颗粒分数占原油总活度的80% 匀浆。这些结果证实了RcGIP1 CDNA编码一个 胰岛素敏感膜相关CGI PDE。目前工作正在进行中。 为了确定观察到的胰岛素刺激是否包括 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.