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

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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.