C-Peptide Binding to Human Cell Membranes: Importance of Glu27

Pramanik, Aladdin; Ekberg, Karin; Zhong, Zhihui; Shafqat, Jawed; Henriksson, Mikael; Jansson, Olof; Tibell, Annika; Tally, Michael; Wahren, John; Jörnvall, Hans; Rigler, Rudolf; Johansson, Jan

doi:10.1006/bbrc.2001.4917

Cited by 59 publications

(60 citation statements)

References 26 publications

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“…Abolition of this binding by pertussis toxin (PTX) was observed, suggesting the involvement of a G-protein coupled receptor (GPCR). The calculated association rate constant (K ass ) of this receptor was about 3 nmol/l, which suggests that under normal physiological conditions it is probably fully occupied, possibly explaining the absence of C-peptide effects in normal subjects [18,19].…”

Section: Introductionmentioning

confidence: 93%

Potent activation of multiple signalling pathways by C-peptide in opossum kidney proximal tubular cells

et al. 2004

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Aims/hypothesis. Proinsulin C-peptide is generally believed to be inert without any appreciable biological functions. However, it has been shown to modulate a variety of cellular processes important in the pathophysiology of diabetic complications. We therefore investigated the ability of C-peptide to stimulate intracellular signalling pathways in kidney proximal tubular cells, the altered activation of which may possibly be related to the development of diabetic nephropathy. Methods. Extracellular signal-regulated kinase (ERK) and Akt phosphorylation were evaluated by western blotting. ERK activity was measured by in vitro kinase assay. Intracellular Ca 2+ was evaluated by confocal imaging. The membrane and cytosol-associated fractions of protein kinase C (PKC) isoforms were evaluated by western blotting. Proliferation was assessed by thymidine incorporation assay. Results. Using the opossum proximal tubular kidney cell line as a model, we demonstrated that at high picomolar to low nanomolar concentrations, C-peptide stimulates extracellular signal-regulated mitogen-activated kinase (3.3±0.1-fold over basal at 3 minutes) and phosphatidylinositol 3-kinase (4.1±0.05-fold over basal at 5 minutes). ERK activation was attenuated by pre-treatment with a PKC inhibitor and abolished by pertussis toxin. Elevations of intracellular [Ca 2+ ] are seen in response to 5 nmol/l C-peptide with consequent activation of PKC-α. Pre-treatment with pertussis toxin abolished PKC-α. C-peptide is also a functional mitogen in this cell type, stimulating significantly increased cell proliferation. Proliferation was attenuated by wortmannin and pertussis toxin pretreatments. None of these effects is reproduced by scrambled C-peptide. Conclusions/interpretation. This study provides evidence that C-peptide, within physiological concentration ranges, stimulates many signalling pathways in opossum kidney cells.

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Section: Introductionmentioning

confidence: 93%

Potent activation of multiple signalling pathways by C-peptide in opossum kidney proximal tubular cells

et al. 2004

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“…Several years ago, C-peptide was shown to specifically bind to plasma membranes from rat pancreatic beta cells [9], human renal tubular cells [10], human fibroblasts and endothelial cells [11]. More recently, C-peptide was shown to bind and cross plasma membranes, localizing in the cytoplasm of HEK-293 cells and Swiss 3T3 fibroblasts [12], where it was detected up to 1 h after its uptake.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2009

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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.

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“…Mutation into the C-peptide to allow processing by furin may alter receptor binding of C-peptide. A recent paper, however, demonstrated that Glu 27 was crucial for binding and is unaltered in our mutant (Pramanik et al 2001). However, further studies would be necessary to demonstrate effective C-peptide membrane binding.…”

Section: Tetracycline-responsive Insulin Secretion · K T Scougall Andmentioning

confidence: 66%

Tetracycline-regulated secretion of human insulin in a transfected non-endocrine cell line

Scougall¹,

Maltin²,

Shaw³

2003

Journal of Molecular Endocrinology

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Long-term constitutive secretion of insulin by implantation of ex vivo transfected cells such as fibroblasts or myoblasts or in situ by intramuscular injection of naked plasmid DNA provides a potential approach to gene therapy for diabetes mellitus. A mechanism for regulating insulin secretion will be necessary to realize the therapeutic potential of this approach. A second obstacle is the inability of non-endocrine host cells to fully process proinsulin. Therefore, alteration of the wild-type cDNA will be necessary to achieve processing of proinsulin by endogenous endoproteases within these cells. The cDNAs for β-galactosidase (β), human wild-type proinsulin (hppI1) and a mutated construct (hppI4), in which the dibasic PC2 and PC3 cleavage sites had been altered to form furin cleavage sites, were sub-cloned into four vectors (pCR3, pVR1012, pIRES, pTRE), including a tetracycline responsive plasmid (pTRE) that requires co-transfection with another plasmid encoding a transactivator (pTet-off) for transgene expression. Transient transfection of the COS-7 fibroblast cell line with these constructs was performed using DEAE-dextran and liposomes. Analysis of vector efficiencies revealed that pTRE/pTetoff>pIRES>pCR3>pVR1012. Further analysis demonstrated total pro/insulin secretion of 2·33 ng/10 6 cells/24 h with ≥25% processed to insulin in hppI-1.pTRE/pTet-off-transfected cells compared with 0·39 ng/10 6 cells/24 h and >70% processing in hppI-4.pTRE/pTet-off-transfected cells. In co-transfection studies with pTRE-hppI1/pTet-off and pTRE-hppI4/pTet-off constructs, pro/insulin secretion was inhibited to 65-66% and 36-38% of control (100%) in the presence of 0·01 and 0·1 µg/ml tetracycline respectively over a 24-h incubation period. Furthermore, reversal of tetracycline inhibition was demonstrated for pTRE-hppI1/pTet-off-and pTRE-hppI4/pTet-off-transfected cells. After a 48-h incubation with 1·0 µg/ml tetracycline, total pro/insulin levels were 10 and 14% compared with untreated cells respectively. On tetracycline removal, total proinsulin levels increased and were equivalent to untreated groups 72 h later. In conclusion, regulation of fully processed human insulin secretion has been achieved in a transiently transfected non-endocrine cell line.

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C-Peptide Binding to Human Cell Membranes: Importance of Glu27

Cited by 59 publications

References 26 publications

Potent activation of multiple signalling pathways by C-peptide in opossum kidney proximal tubular cells

Potent activation of multiple signalling pathways by C-peptide in opossum kidney proximal tubular cells

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

Tetracycline-regulated secretion of human insulin in a transfected non-endocrine cell line

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