Human C-peptide antagonises high glucose-induced endothelial dysfunction through the nuclear factor-κB pathway

Abstract

Aims/hypothesis Endothelial dysfunction in diabetes is predominantly caused by hyperglycaemia leading to vascular complications through overproduction of oxidative stress and activation of the transcription factor nuclear factor-κB (NF-κB). Many studies have suggested that decreased circulating levels of C-peptide may play a role in diabetic vascular dysfunction. To date, the possible effects of C-peptide on endothelial cells and intracellular signalling pathways are largely unknown. We therefore investigated the effect of C-peptide on several biochemical markers of endothelial dysfunction in vitro. To gain insights into potential intracellular signalling pathways affected by C-peptide, we tested NF-κB activation, since it is known that inflammation, secondary to oxidative stress, is a key component of vascular complications and NF-κB is a redox-dependent transcription factor. Methods Human aortic endothelial cells (HAEC) were exposed to 25 mmol/l glucose in the presence of C-peptide (0.5 nmol/l) for 24 h and tested for expression of the gene encoding vascular cell adhesion molecule-1 (VCAM-1) by RT-PCR and flow cytometry. Secretion of IL-8 and monocyte chemoattractant protein-1 (MCP-1) was measured by ELISA. NF-κB activation was analysed by immunoblotting and ELISA. Results Physiological concentrations of C-peptide affect high glucose-induced endothelial dysfunction by: (1) decreasing VCAM-1 expression and U-937 cell adherence to HAEC; (2) reducing secretion of IL-8 and MCP-1; and (3) suppressing NF-κB activation. Conclusions/interpretation During hyperglycaemia, Cpeptide directly affects VCAM-1 expression and both MCP-1 and IL-8 HAEC secretion by reducing NF-κB activation. These effects suggest a physiological anti-inflammatory (and potentially anti-atherogenic) activity of C-peptide on endothelial cells.

C-peptide is internalised in human endothelial and vascular smooth muscle cells via early endosomes

“…The effect of C-peptide that we observed on the nuclear factor kappaB (NF-κB) pathway in both HAEC and UASMC [13,15] might thus originate from C-peptidereceptor complex signalling from the endosomes, as has been demonstrated for certain Toll-like receptor pathways and other inflammatory pathways, which affect the activation of the NF-κB pathway from the endosomes [34,35]. The mildly acidic pH of the sorting endosomes would then begin the dissociation of the C-peptide destined to lysosomes from its recycled receptor.…”

“…Recent studies have, however, challenged this classical view by demonstrating that C-peptide is capable of biological effects in many different cell types and, importantly, reduces the complications associated with type 1 diabetes [2,[5][6][7][8]. Previous work from our laboratory has demonstrated a protective effect of C-peptide on the vasculature in hyperglycaemic conditions by decreasing inflammatory damage and the activation of nuclear factor (NF)-κB [13,15]. More recently, a physiological role in glucose clearance and the release of ATP from erythrocytes of diabetic patients has been suggested [27].…”

“…The internalisation of C-peptide was explored in human aortic endothelial cells (HAEC) and umbilical artery smooth muscle cells (UASMC), two important targets of C-peptide activity, especially in the context of vascular dysfunction leading to vascular complications in type 1 diabetes [13][14][15][16]. We found that in these cells C-peptide internalises from the cellular surface within punctate structures, most of them co-localizing with early endosomes.…”

C-peptide reduces high-glucose-induced apoptosis of endothelial cells and decreases NAD(P)H-oxidase reactive oxygen species generation in human aortic endothelial cells

“…As type 1 diabetes patients typically lack physiological levels of insulin and C-peptide, this is considered an important factor in the pathophysiology of diabetic complications [24][25][26]. C-peptide has been shown to improve endothelial dysfunction and systemic inflammation in several in vivo and in vitro models of inflammation-mediated vascular injury by reducing expression of genes encoding endothelial cell adhesion molecules, inflammatory cytokine production and adherence and transmigration of leucocytes [27][28][29][30]. Although the exact mechanism(s) underlying the antiinflammatory activity of C-peptide is not known, there is evidence that C-peptide affects NF-κB activation [29,31].…”

“…C-peptide has been shown to improve endothelial dysfunction and systemic inflammation in several in vivo and in vitro models of inflammation-mediated vascular injury by reducing expression of genes encoding endothelial cell adhesion molecules, inflammatory cytokine production and adherence and transmigration of leucocytes [27][28][29][30]. Although the exact mechanism(s) underlying the antiinflammatory activity of C-peptide is not known, there is evidence that C-peptide affects NF-κB activation [29,31]. However, which NF-κB-dependent upstream signalling event is affected by C-peptide in endothelial cells is not clear.…”