Potent activation of multiple signalling pathways by C-peptide in opossum kidney proximal tubular cells |
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Authors: | N?M?Al-Rasheed F?Meakin E?L?Royal A?J?Lewington J?Brown G?B?Willars Email author" target="_blank">N?J?BrunskillEmail author |
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Institution: | (1) Department of Cell Physiology and Pharmacology, Faculty of Medicine and Biological Sciences, University of Leicester, Medical Sciences Building, University Road, Leicester, LE1 9HN, United Kingdom;(2) Department of Nephrology, Faculty of Medicine and Biological Sciences, University of Leicester, Leicester, UK;(3) Department of Pharmacology, King Saud University, Riyadh, Saudi Arabia |
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Abstract: | 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 Ca2+ 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 Ca2+] are seen in response to 5 nmol/l C-peptide with consequent activation of PKC- . Pre-treatment with pertussis toxin abolished PKC- . C-peptide is also a functional mitogen in this cell type, stimulating significantly increased cell proliferation. Proliferation was attenuated by wortmannin and pertussis toxin pre-treatments. 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.Abbreviations Akt/PKB
protein kinase B
- DTT
dithiothreitol
- ERK
extracellular signal-regulated kinase
- GPCR
G-protein coupled receptor
- MAPK
mitogen-activated protein kinase
- OK
opossum kidney
- PI3-K
phosphoinositide 3-kinase
- PKC
protein kinase C
- PMA
phorbol myristate acetate
- PMSF
phenylmethansulfonylfluoride
- PTC
proximal tubular cells
- PTX
pertussis toxin |
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Keywords: | Calcium C-peptide Diabetic nephropathy Kidney MAP kinase Mitogen PI 3-kinase PKC |
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