C-peptide enhances insulin-mediated cell growth and protection against high glucose–induced apoptosis in SH-SY5Y cells

Abstract

Background

We have previously reported that C-peptide exerts preventive and therapeutic effects on diabetic neuropathy in type 1 diabetic BB/Wor-rats and that it prevents duration-dependent hippocampal apoptosis in the same animal model. In the present study, we examined human neuroblastoma SH-SY5Y cells to examine whether C-peptide stimulates cell proliferation/neurite outgrowth and whether it has antiapoptotic effects.

Methods

For neurite outgrowth, serum-starved cultures were treated with C-peptide and/or insulin or IGF-1. Neurites were visualized with NF-L antibody and measured morphometrically. Cell numbers were determined using an electronic cell counter. Scrambled C-peptide was used as a negative control. For assessment of apoptosis, SH-SY5Y cells were incubated with 100 mM glucose for 24 h, and the effects of C-peptide and/or insulin or IGF-1 were examined. Apoptosis was demonstrated by transferase-mediated dUTP nick-end labeling (TUNEL)/4,6-diamidino-2-phenylindole (DAPI) stainings, flow cytometry and changes in the expression of Bcl₂. Activation of insulin signaling intermediaries was determined by Western blots. Translocation of NF-κB was demonstrated immunocytochemically.

Results

C-peptide but not scrambled C-peptide stimulated cell proliferation and neurite outgrowth. In the presence of 4 nM insulin, 3 nM C-peptide significantly increased autophosphorylation of the insulin receptor (IR) but not that of the insulin-like growth factor 1 receptor (IGF-1R). It stimulated phosphoinositide 3-kinase (PI-3 kinase) and p38 mitogen-activated protein (MAP) kinase activation, enhanced the expression and translocation of nuclear factor-κB (NF-κB), promoted the expression of Bcl₂ and reduced c-jun N-terminal kinase (JNK) phosphorylation in excess of that of insulin alone.

Conclusions

C-peptide in the presence of insulin exerts synergistic effects on cell proliferation, neurite outgrowth and has in the presence of insulin an antiapoptotic effect on high glucose–induced apoptosis but less so on hyperosmolar-induced apoptosis. These effects are likely to be mediated via interactions with the insulin signaling pathway. Copyright © 2003 John Wiley & Sons, Ltd.