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Diabetologia

, Volume 44, Issue 10, pp 1281–1295 | Cite as

NGF-withdrawal induces apoptosis in pancreatic beta cells in vitro

  • D. Pierucci
  • S. Cicconi
  • P. Bonini
  • F. Ferrelli
  • D. Pastore
  • C. Matteucci
  • L. Marselli
  • P. Marchetti
  • F. Ris
  • P. Halban
  • J. Oberholzer
  • M. Federici
  • F. Cozzolino
  • R. Lauro
  • P. Borboni
  • L. N. J. L. Marlier
Article

Abstract.

Aims/hypothesis:

Using primary cultures of human pancreatic islets, purified human pancreatic beta cells and the mouse βTC6-F7 cell line, we analysed the expression of nerve growth factor, (NGF/NGF) receptors in beta cells. To investigate whether NGF could sub-serve an autocrine antiapoptotic role in beta cells, we studied the effects of NGF withdrawal using a neutralizing monoclonal anti-NGF antibody.

Methods:

The expression of NGF and NGF receptors (gp140Trk-A and p75NTR) were analysed by RT-PCR and immunofluorescence. Pulse-chase experiments and beta cell/PC12 co-cultures were used to investigate NGF production and secretion from beta cells. Possible apoptosis induced by NGF withdrawal was monitored by phosphatidylserine translocation, nucleosomal formation, DNA laddering and FACS analysis. Involvement of transcription/translation mechanisms were investigated as well as the gp140Trk-A required. Finally, signal transduction pathways typically involved in apoptotic mechanisms were analysed by western blot analysis.

Results:

We show that NGF and both NGF receptors, gp140Trk-A and p75NTR are expressed in beta cells where NGF is produced and secreted in a biologically active form. NGF-withdrawal induces beta-cell transcription/translation independent apoptosis but mediated by gp140Trk-A. Analysis of signal transduction pathways revealed that NGF withdrawal inhibits the PI3-K, protein kinase B (AKT), Bad survival pathway and activates c-Jun kinase (JNK) whereas ERKs and p38 mitogen-activated protein kinase (MAPK) are not affected. Moreover, Bcl-XL, but not Bcl-2 protein expression are reduced.

Conclusion/interpretaiton:

We suggest that the integrity of the NGF/NGF receptor system and NGF bioavailability participate in controlling beta-cell survival in culture which represents a key issue for improving possibilities for transplantations in the treatment of diabetes. [Diabetologia (2001) 44: 1281–1295]

Keywords Islet cell nerve growth factor nerve growth factor receptors diabetes mellitus apoptosis cell death protein-tyrosine kinase proto-oncogene proteins c-bcl-2 signal transduction phosphorylation. 

Copyright information

© Springer-Verlag Berlin Heidelberg 2001

Authors and Affiliations

  • D. Pierucci
    • 1
  • S. Cicconi
    • 1
  • P. Bonini
    • 1
  • F. Ferrelli
    • 1
  • D. Pastore
    • 1
  • C. Matteucci
    • 2
  • L. Marselli
    • 3
  • P. Marchetti
    • 3
  • F. Ris
    • 4
  • P. Halban
    • 4
  • J. Oberholzer
    • 5
  • M. Federici
    • 1
  • F. Cozzolino
    • 3
  • R. Lauro
    • 1
  • P. Borboni
    • 1
  • L. N. J. L. Marlier
    • 1
  1. 1.Laboratory Molecular Medicine, Department of Internal Medicine, University of Rome “Tor Vergata”, Rome, ItalyIT
  2. 2.Department of Experimental Medicine and Biochemical Sciences,University of Rome “Tor Vergata”, ItalyIT
  3. 3.Department of Endocrinology and Metabolism, University of Pisa, ItalyIT
  4. 4.Louis-Jeantet Research Laboratories, University Medical Center, Geneva, SwitzerlandCH
  5. 5.Division of Surgical Investigation, Clinic of Digestive Surgery, Geneva, SwitzerlandCH

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