Proinsulin C-peptide increases nitric oxide production by enhancing mitogen-activated protein-kinase-dependent transcription of endothelial nitric oxide synthase in aortic endothelial cells of Wistar rats

Aims/hypothesis. Recent studies have suggested that proinsulin C-peptide improves vascular functions, possibly through nitric oxide (NO) production. To clarify the molecular mechanisms of vascular NO production induced by C-peptide, we examined the effects of C-peptide on NO production and NO synthase expression in rat aortic endothelial cells in connection with mitogen-activated protein kinase (MAPK) activation. Methods. Aortic endothelial cells were isolated from female Wistar rats, cultured to confluence, and serum-starved for 24 h before treatment with C-peptide. Nitric oxide production was measured by the DAF-2 fluorescence dye method and relative amounts of endothelial nitric oxide synthase (eNOS) protein and its mRNA were semi-quantified by western blot and RT-PCR analyses respectively. Activation of MAPK was estimated by western blot detection of activity-related phosphorylation and in vitro kinase assay. Results. Stimulation of cells with C-peptide for 3 h doubled NO production, which was suppressed by the NO synthase inhibitor, N^G-nitro-L- arginine methyl ester (L-NAME). Stimulation also increased mRNA and protein contents of eNOS in a manner sensitive to the transcription inhibitor actinomycin D. It did not affect inducible NO synthase mRNA. C-peptide also induced rapid phosphorylation and activation of extracellular signal-regulated kinase (ERK, also known as p44/42MAPK), but not of p38MAPK. In cells pretreated with the ERK inhibitor PD98059 the C-peptide-elicited increase of NO production and eNOS was abrogated in a dose-dependent manner; suppression of ERK phosphorylation induced by C-peptide also occurred. Conclusions/interpretation. Our results show that C-peptide increases NO production by increasing eNOS protein contents through ERK-dependent up-regulation of eNOS gene transcription. This could explain some actions of C-peptide on the vasculature, indicating a pivotal role for C-peptide in vascular homeostasis.