Identification of novel adipocyte-specific factors regulating PDE3B gene transcription different from PPAR_γ

磷酸二酯酶3B（PDE3B）可被胰岛素激活，从而抑制脂肪分解并减少脂肪细胞的游离脂肪酸输出。特异性增强PDE3B基因表达可改善胰岛素抵抗。我们计划通过分析缺乏PPAR结合位点的PDE3B基因启动子来鉴定不同于PPAR_γ的调节PDE3B基因转录的新的脂肪细胞特异性因子。我们首先构建了从~ 2kb长度开始的5'缺失启动子报告子。同时，我们也在人类PDE3B基因启动子中未发现PPAR结合位点。然后我们分析了人类PDE3B基因启动子。我们分离了PDE3B基因的5'侧翼区域，并通过引物延伸确定了转录起始位点。然后，我们使用PCR直接测序方法在24名日本2型糖尿病患者的5'侧区搜索这2kb的多态性，并对包含已鉴定多态性的区域进行检测。只有-465G>T和- 1727_l726instcaatt的频率超过5%。由于两者之间存在完全的连锁不平衡，我们进一步分析了-465G>T，以及之前在编码区发现的+1389G> a，共200例对照和207例2型糖尿病患者。这些snp和单倍型与2型糖尿病无关。T/T基因型-465是罕见的，尽管2型糖尿病患者（4/207名受试者）的这一频率可能高于对照组（0/200名受试者）。因此，确定的多态性不太可能对日本2型糖尿病的易感性产生重大影响。我们已经构建了人类PDE3B基因启动子报告子。我们正在使用小鼠缺失构建体和人类构建体进行荧光素酶检测。我们还研究了鉴定的多态性对蛋白质与DNA元件结合的影响，使用电泳迁移位移测定。

Phosphodiesterase 3B(PDE3B) is activated by insulin, which inhibits lipolysis and reduces free fatty acid output from adipocytes. Specific enhancement of PDE3B gene expression could improve insulin resistance. We planned to identify novel adipocyte-specific factors regulating PDE3B gene transcription different from PPAR_γ, by analyzing PDE3B gene promoter lacking PPAR binding sites.We first constructed 5' deletion promoter reporters starting from the〜2kb length. In parallel, we also found no PPAR binding sites in the human PDE3B gene promoter. We then analyzed the human PDE3B gene promoter. We isolated the 5' flanking region of the PDE3B gene and determined the transcription initiation site by primer extension. We then searched for polymorphisms in this 2kb of the 5' flanking region in 24 type 2 diabetic Japanese subjects using PCR direct sequencing, and the regions including the identified polymorphisms were then examined. Only -465G>T and -1727_-l726insTCAATT had more than 5% frequencies. Since a complete linkage disequilibrium existed between them, -465G>T was further analyzed, along with a previously identified +1389G>A in the coding region in a total of 200 controls and 207 type 2 diabetic subjects. These SNPs and haplotypes were not associated with type 2 diabetes. The T/T genotype at -465 was rare although this frequency could be higher in type 2 diabetes(4/207 subjects) than controls(0/200 subjects). Thus, the identified polymorphisms are unlikely to have major effects on susceptibility to Japanese type 2 diabetes.We have already constructed human PDE3B gene promoter reporters. We are doing luciferase assays using mouse deletion constructs and human constructs. We are also examining th~ effects of the identified polymorphisms on the protein binding to the DNA elements using electrophoretic mobility shift assays.