Negative regulation of insulin-induced tetrahydrobiopterin synthesis by protein kinase C in vascular endothelial cells

We examined the role of protein kinase C (PKC) in the insulin-induced biosynthesis of tetrahydrobiopterin (BH4), which is one of the cofactors of nitric oxide (NO) synthase (NOS), in vascular endothelial cells. The BH4 level was determined as biopterin by reversed-phase high performance liquid chromatography with fluorometric detection. Measurement of the level of mRNA for GTP cyclohydrolase I (GTPCH), which is the first and the rate-limiting enzyme for the novo pathway for BH4 synthesis, was performed by reverse transcription-polymerase chain reaction (RT-PCR). Treatment with insulin increased the BH4 level and the GTPCH mRNA level in endothelial cells. The insulin-induced increases of both GTPCH mRNA and BH4 levels were enhanced by co-treatment with bisindolylmaleimide I or Ro 31-8220, inhibitors of PKC. On the other hand, the insulin-induced increases of BH4 synthesis and expression of GTPCH mRNA were also enhanced by co-incubation with a PKC activator, phorbol 12-myristate 13-acetate (PMA) for 24 h. Long-term treatment with PMA is known to down-regulate PKC. Addition of insulin after pretreatment with PMA for 24 h markedly enhanced BH4 synthesis and expression of GTPCH mRNA. These findings suggest that PKC negatively regulates the induction of BH4 synthesis by insulin endothelial cells. Insulin-induced BH4 synthesis may be suppressed in diabetes mellitus, since PKC has been shown to be up-regulated in diabetes mellitus.