C-peptide is internalised in human endothelial and vascular smooth muscle cells via early endosomes.
OBJECTIVE
There is increasing evidence that C-peptide 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 C-peptide 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 degrees 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
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.
Luppi
P
P
Department of Pediatrics, Children's Hospital of Pittsburgh of UPMC, Rangos Research Center, PA 15201, USA. luppip@pitt.edu
Geng
X
X
Cifarelli
V
V
Drain
P
P
Trucco
M
M
D000840:Anisoles / Q000494:pharmacology; D002096:C-Peptide / Q000378:metabolism*; D002460:Cell Line; D003508:Cycloheptanes / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism*; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D005372:Filipin / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism*; D015739:Nocodazole / Q000494:pharmacology; D013696:Temperature
C109046:2-methoxy-5-(2',4'-dimethoxyphenyl)-2,4,6-cycloheptatrien-1-one; D000840:Anisoles; D002096:C-Peptide; D003508:Cycloheptanes; D015638:Cytochalasin D; D005372:Filipin; D015739:Nocodazole