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

Abstract

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 Cpeptide 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. [Diabetologia (2003[Diabetologia ( ) 46:1698[Diabetologia ( -1705

C-peptide stimulates ERK1/2 and JNK MAP kinases via activation of protein kinase C in human renal tubular cells

“…It has been suggested that C-peptide plays an important role in the maintenance of vascular homeostasis via its effects on eNOS [8]. There is also evidence to indicate that C-peptide participates in the control of renal Na + , K + -ATPase activity, thereby contributing to the regulation of tubular sodium handling during postprandial periods [6,7].…”

“…There is evidence to suggest that C-peptide binds to a G-protein-coupled membrane binding site on a number of different cell types [1], thereby triggering Ca 2+ -dependent intracellular signalling pathways [2] including the mitogen-activated protein (MAP) kinase cascade [3,4]. This results in subsequent activation of both Na + , K + -ATPase [5][6][7] and endothelial nitric oxide synthase (eNOS) [5][6][7][8]. Activation of these enzyme systems is of particular interest in diabetes, since both are reported to be deficient in this disorder [9][10][11][12].…”

“…It increases the PKC-dependent phosphorylation of ERK1/2 in Swiss 3T3 fibroblasts [3] and in opossum renal tubular cells [26]. C-peptide also stimulates the phosphorylation of ERK1/2 and p38 MAP kinase in endothelial cells [27], resulting in increased eNOSmediated synthesis of NO and augmented formation of eNOS protein, secondary to upregulation of eNOS gene transcription, in rat aortic endothelial cells [8]. Moreover, C-peptide stimulates Na + , K + -ATPase activity [6] via PKC-α activation in rat medullary thick ascending limb cells [7].…”

C-Peptide Replacement Therapy and Sensory Nerve Function in Type 1 Diabetic Neuropathy

“…Accompanying these beneficial effects of C-peptide is a partial restoration of the diabetesinduced reduction in Na ϩ ,K ϩ -ATPase activity of the nerves (13,14). Moreover, C-peptide is known to have a stimulatory effect on endothelial nitric oxide synthase (15,16), thereby augmenting endoneurial blood flow (7,17). C-peptide also exerts neurotrophic effects and has an inhibitory effect on cellular apoptosis (for review, see ref.…”

C-peptide increases the expression of vasopressin-activated calcium-mobilizing receptor gene through a G protein-dependent pathway

“…Activation of the Na -K pump by C-peptide was also demonstrated in man (15), but whether the protection by C-peptide against diabetic complications is mediated by this mechanism remains unclear. Of interest, recent findings indicate that C-peptide increases calcium influx and stimulates endothelial nitric oxide synthase (16,17), possibly through the activation of the mitogen-activated protein (MAP) kinase (18).…”