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

T. Kitamura, K. Kimura, K. Makondo, D. T. Furuya, M. Suzuki, T. Yoshida, M. Saito

Research output: Contribution to journalArticle

85 Scopus citations


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, NG-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.

Original languageEnglish (US)
Pages (from-to)1698-1705
Number of pages8
Issue number12
StatePublished - Dec 1 2003
Externally publishedYes



  • Actinomycin D
  • C-peptide
  • ERK
  • Endothelial cells
  • L-NAME
  • MAPK
  • Nitric oxide
  • Proinsulin
  • eNOS
  • p38MAPK

ASJC Scopus subject areas

  • Internal Medicine
  • Endocrinology, Diabetes and Metabolism

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