Stimulation of tetrahydrobiopterin synthesis induced by insulin: possible involvement of phosphatidylinositol 3-kinase.

Although we recently showed that insulin increases the intracellular concentration of tetrahydrobiopterin (BH4), which is one of the cofactors of nitric oxide (NO) synthase, the mechanism of the effect was not elucidated. In the present study, we examined the signaling pathway of the stimulation of BH4 synthesis by insulin in mouse brain microvascular endothelial cells. Extracellular and intracellular BH4 levels were determined as biopterin by using reversed-phase high performance liquid chromatography with fluorometric detection. Measurement of the level of mRNA for GTP cyclohydrolase I (GTPCH), which is the rate-limiting enzyme for de novo BH4 synthesis, was performed by reverse transcription-polymerase chain reaction (RT-PCR). Addition of insulin to endothelial cells caused an increase of not only the intracellular but also the extracellular BH4 level in a time- and a concentration-dependent manner. Insulin also induced an increase of the level of GTPCH mRNA. Moreover, 2,4-diamino-6-hydroxypyrimidine, an inhibitor of GTPCH, inhibited the insulin-induced enhancement of BH4 synthesis. The increase in the BH4 level and the induction of GTPCH mRNA by insulin were reduced by wortmannin and LY294002, which are both phosphatidylinositol 3-kinase (PI3-kinase) inhibitors. These results suggest that insulin stimulates BH4 synthesis through the de novo synthetic pathway involving induction of GTPCH, and that the signaling pathway involves the activation of PI3-kinase.