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

Although we recently showed that insulin increases the intracellular concen tration of tetrahydrobiopterin (BH4), which is one of the cofactors of nitr ic 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. Ex tracellular and intracellular BH4 levels were determined as biopterin by us ing reversed-phase high performance liquid chromatography with fluorometric detection. Measurement of the level of mRNA for GTP cyclohydrolase I (GTPC H), which is the rate-limiting enzyme for de novo BH4 synthesis, was perfor med by reverse transcription-polymerase chain reaction (RT-PCR). Addition o f insulin to endothelial cells caused an increase of not only the intracell ular but also the extracellular BH4 level in a time- and a concentration-de pendent manner. Insulin also induced an increase of the level of GTPCH mRNA . Moreover, 2,4-diamino-6-hydroxypyrimidine, an inhibitor of GTPCH, inhibit ed the insulin-induced enhancement of BH4 synthesis. The increase in the BH 4 level and the induction of GTPCH mRNA by insulin were reduced by wortmann in and LY294002, which are both phosphatidylinositol 3-kinase (PI3-kinase) inhibitors. These results suggest that insulin stimulates BH4 synthesis thr ough the de novo synthetic pathway involving induction of GTPCH, and that t he signaling pathway involves the activation of PI3-kinase. (C) 2001 Elsevi er Science Ltd. All rights reserved.