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.
OBJECTIVE
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
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.
Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan. k-kimura@vetmed.hokudai.ac.jp
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Kitamura
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Kimura
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Makondo
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Furuya
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Suzuki
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Yoshida
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Saito
D000818:Animals; D001011:Aorta; D001483:Base Sequence; D002096:C-Peptide / Q000494:pharmacology*; D002478:Cells, Cultured; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D005260:Female; D007700:Kinetics; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052250:Nitric Oxide Synthase Type III; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics*
D002096:C-Peptide; D017931:DNA Primers; D012333:RNA, Messenger; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D020928:Mitogen-Activated Protein Kinases