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

Abstract

Aims/hypothesis There is increasing evidence that Cpeptide exerts intracellular effects in a variety of cells and could be beneficial in patients with type 1 diabetes. Exactly how C-peptide achieves these effects, however, is unknown. Recent reports showed that C-peptide internalised in the cytoplasm of HEK-293 and Swiss 3T3 cells, where it was not degraded for at least 1 h after uptake. In this study, we investigated the hypothesis that C-peptide is internalised via an endocytic pathway and traffics to classic endocytic organelles, such as endosomes and lysosomes. Methods We studied the internalisation of C-peptide in vascular endothelial and smooth muscle cells, two relevant targets of Cpeptide activity, by using Alexa Fluor-labelled C-peptide probes in living cells and immunohistochemistry employing confocal laser-scanning microscopy. To examine trafficking to subcellular compartments, we used fluorescent constructs tagged to RAB5A, member RAS oncogene family (RAB5A) to identify early endosomes, or to lysosomal-associated membrane protein 1 (LAMP1) to identify lysosomes.Results C-peptide internalised in the cytoplasm of cells within punctate structures identified as early endosomes. Internalisation was clearly detectable after 10 min of incubation and was blocked at 4°C as well as with excess of unlabelled C-peptide. A minor fraction of vesicles, which increased with culture time, co-localised with lysosomes. Uptake of C-peptide was reduced by monodansylcadaverine, a pharmacological compound that blocks clathrin-mediated endocytosis, and by nocodazole, which disrupts microtubule assembly. Conclusions/interpretation C-peptide internalises in the cytoplasm of cells by endocytosis, as demonstrated by its localisation in early endosomes. Endosomes might represent a signalling station, through which C-peptide might achieve its cellular effects.

Molecular Effects of C-Peptide in Microvascular Blood Flow Regulation

“…Recent data suggest that C-peptide binds to a G-protein coupled receptor, increasing Ca 2+ influx, with subsequent activation of Ca 2+ -calmodulin. In human aortic endothelial cells, and in umbilical artery smooth muscle cells, C-peptide was shown to be internalized in the cell by endocytosis [63]. Binding of Cpeptide to a specific membrane receptor with consecutive endocytosis might represent a conceivable signaling pathway to achieve the stimulation of eNOS or Na + /K + -ATPase in erythrocytes and endothelial cells.…”

Physiological effects and therapeutic potential of proinsulin C-peptide

“…By reducing excessive ROS accumulation in endothelial cells exposed to high glucose or other stressful agents, C-peptide inhibits the generation of a cascade of deleterious inflammatory responses that eventually results in cellular death. The specific intracellular pathways by which C-peptide achieves its anti-inflammatory effects in target cells after binding to cellular membranes and localization to early endosomes are largely unknown (66). As discussed below, evidence for a putative G protein-coupled receptor (GPCR) for C-peptide, GPR146, has been recently reported for different cell types (113).…”

“…In addition, C-peptide has been shown to stimulate extracellular signal-related kinase (ERK) activity, intracellular calcium accumulation, and protein kinase C (PKC) activity (1,118,119). C-peptide has been shown to be internalized by human endothelial cells (66) and to reduce endothelial dysfunction via the NF-B pathway (64). Additionally, C-peptideinduced p38 MAPK activation was shown to stimulate the production of c-Jun NH 2 -terminal kinase (JNK) in lung endothelial cells (28).…”

Breakthrough in Diabetes Therapy ... Just Around the Corner?

“…In endothelial and vascular smooth muscle cells exposed to hyperglycemia, C-peptide is internalized via early endosomes. Once inside the cell, it can reduce inflammatory damage mediated by nuclear factor (NF)-κB activation and upregulation of RAGE signaling [14,15]. Beside relieving states of glucose instability and problems with hypoglycemia, islet transplantation promotes C-peptide secretion.…”