Expression, Structure/function And Regulation Of Phospho

环核苷酸磷酸二酯酶（PDEs）是cAMP和cGMP细胞内浓度和生物学效应的关键调节因子。了解PDE亚型[属于11个基因家族（PDEs 1-11）]的细胞表达和调控对于靶向特异性PDE治疗包括肺部疾病在内的各种疾病越来越重要。PDE3B在培养的人脂肪细胞、小鼠3T3-L1脂肪细胞或人单核细胞源性巨噬细胞的分化过程中出现（或活性大幅增加）。我们的研究结果表明，cAMP（在IBMX反应中积累）在3T3-L1脂肪细胞分化过程中对PDE3B表达的调节中起着至关重要的作用，而PDE3B的诱导似乎与脂质积累没有直接联系。我们的研究结果还表明，胰岛素诱导的PDE3B的激活是由PI3-K-和PKB依赖性信号介导的，并且可能与IRS-1诱导的信号分子易位到细胞膜内有关，PDE3B可能被PKB激活。此外，在溶解3T3-L1微粒体膜的凝胶过滤层析过程中，发现PKB细胞内池的一部分与细胞膜相关，与膜相关的PDE3B共洗脱，并与PDE3B共免疫沉淀。后一种相互作用的结构决定因素位于PDE3B的n端调控区域和PKB的ph结构域；来自n端区域的富含脯氨酸的肽序列似乎抑制PDE3B和PKB之间的相互作用。为了进一步研究PDE3亚型的功能作用，我们产生了PDE3A和PDE3B基因靶向破坏的小鼠。雌性PDE3A KO小鼠是不育的，很可能是因为卵母细胞中缺乏功能性PDE3A导致cAMP增加，蛋白激酶A （PKA）的激活，以及成熟促进因子/ map激酶信号的抑制，这些信号在减数分裂过程、卵母细胞成熟（以生发囊泡破裂（GVBD）为标志）和随后的受精中很重要。与这一假设一致，PKA抑制剂处理PDE3A KO卵母细胞重新启动减数分裂进程，并恢复体外受精能力。PDE3B KO小鼠似乎积累较少的脂肪，并表现出胰岛素稳态机制破坏和胰岛素抵抗的迹象。对完整小鼠施用β -3激动剂导致PDE3B KO小鼠血清胰岛素大幅增加，但相对较少的葡萄糖处理，这也显示出与血糖降低有关的异常胰岛素耐量试验。高胰岛素-正糖钳表明胰岛素在抑制KO小鼠内源性葡萄糖产生方面的效果要差得多。研究正在进行中，以了解PDE3A和PDE3B在这些和其他表型变化中的作用。

Cyclic nucleotide phosphodiesterases (PDEs) are critical regulators of intracellular concentrations and biological effects of cAMP and cGMP. Understanding cellular expression and regulation of PDE isoforms [which belong to eleven gene families (PDEs 1-11)] will be of increasing importance for targeting specific PDEs in treating various diseases, including pulmonary disorders. PDE3B appears (or greatly increases in activity) during differentiation of cultured human adipocytes, murine 3T3-L1 adipocytes, or human monocyte-derived macrophages. Our results suggest that cAMP (accumulating in response to IBMX) plays a crucial role in regulation of PDE3B expression during differentiation of 3T3-L1 adipocytes, and induction of PDE3B does not seem to be directly linked to accumulation of lipid. Our results also indicate that insulin-induced activation of PDE3B is mediated by PI3-K- and PKB-dependent signals, and may be related to IRS-1 induced translocation of signaling molecules to intracellular membranes, where PDE3B may be activated by PKB. In addition a portion of the intracellular pool of PKB is found in association with intracellular membranes, co-elutes with membrane-associated PDE3B during gel filtration chromatography of solubilized 3T3-L1 microsomal membranes, and co-immunoprecipitates with PDE3B. The structural determinants for this latter interaction reside in the N-terminal regulatory region of PDE3B and PH-domain of PKB; proline-rich peptide sequences from the N-terminal region seem to inhibit interactions between PDE3B and PKB. To further examine functional roles of PDE3 isoforms, mice with targeted disruptions of PDE3A and PDE3B genes have been generated. Female PDE3A KO mice are sterile, most likely because the absence of functional PDE3A in oocytes leads to increased cAMP, activation of Protein kinase A (PKA), and inhibition of Maturation-promoting factor/ MAP-kinase signals that are important in meiotic progression , oocyte maturation (marked by germinal vesicle breakdown (GVBD)), and subsequent fertilization. Consistent with this hypothesis, treatment of PDE3A KO oocytes with PKA inhibitors reinitiates meiotic progression, and restores competency for fertilization in vitro. PDE3B KO mice seem to accumulate less fat and exhibit signs of disruption of insulin homeostatic mechanisms and insulin resistance. Administration of a Beta-3 agonist to intact mice results in a much larger increase in serum insulin but relatively less glucose disposal in PDE3B KO mice, which also demonstrate aberrant i.p. insulin tolerance tests with respect to reduction in blood glucose. Hyperinsulinemic-euglycemic clamps indicate that insulin is much less effective in inhibiting endogenous glucose production in KO mice. Studies are ongoing to understand the roles of PDE3A and PDE3B in these and other phenotypic changes.