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

Diabetologia. 2004 Jun;47(6):987-97. doi: 10.1007/s00125-004-1404-9. Epub 2004 May 26.

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 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-alpha. Pre-treatment with pertussis toxin abolished PKC-alpha. 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Androstadienes / pharmacology
  • Animals
  • Blotting, Western / methods
  • C-Peptide / chemistry
  • C-Peptide / pharmacology
  • C-Peptide / physiology*
  • Calcium / metabolism
  • Cell Line
  • Cell Proliferation / drug effects
  • Extracellular Signal-Regulated MAP Kinases / antagonists & inhibitors
  • Extracellular Signal-Regulated MAP Kinases / drug effects
  • Extracellular Signal-Regulated MAP Kinases / physiology*
  • Humans
  • Indoles / pharmacology
  • Kidney Tubules, Proximal / cytology*
  • Kidney Tubules, Proximal / drug effects
  • Kidney Tubules, Proximal / metabolism
  • Microscopy, Confocal / methods
  • Opossums
  • Pertussis Toxin / pharmacology
  • Phosphorylation / drug effects
  • Protein Kinase C / antagonists & inhibitors
  • Protein Kinase C / drug effects
  • Protein Kinase C / metabolism
  • Protein Kinase C-alpha
  • Protein-Serine-Threonine Kinases / drug effects
  • Protein-Serine-Threonine Kinases / metabolism
  • Proto-Oncogene Proteins / drug effects
  • Proto-Oncogene Proteins / metabolism
  • Proto-Oncogene Proteins c-akt
  • Thymidine / metabolism
  • Tritium / metabolism
  • Wortmannin

Substances

  • Androstadienes
  • C-Peptide
  • Indoles
  • Proto-Oncogene Proteins
  • Tritium
  • Pertussis Toxin
  • AKT1 protein, human
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-akt
  • PRKCA protein, human
  • Protein Kinase C
  • Protein Kinase C-alpha
  • Extracellular Signal-Regulated MAP Kinases
  • Calcium
  • Thymidine
  • Ro 31-8220
  • Wortmannin