Notch signalling suppresses apoptosis in adult human and mouse pancreatic islet cells.
OBJECTIVE
The pathogenesis of diabetes and the success of islet transplantation depend on the control of pancreatic beta cell fate. The Notch signalling pathway is essential for normal prenatal pancreatic development, but the presence and function of this gene network in adult islets has received much less attention.


METHODS
The presence of Notch signalling components was assessed in vitro using RT-PCR, western blotting and immunofluorescence. The functional consequences of altering Notch signalling on insulin secretion and programmed cell death were examined.


RESULTS
Adult mouse islets, human islets and mouse insulinoma MIN6 cells possess key components of the Notch pathway. RT-PCR, western blotting and immunofluorescence indicated that the Notch target gene, neurogenin3 (Ngn3, also known as Neurog3), is also present in adult islet cells. Inhibiting Notch signalling with N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT) increased Ngn3 mRNA expression and protein levels in adult islets. The activated notch homologue 1 (NOTCH1) protein level was decreased upon serum withdrawal, as well as after treatment with a phosphatidylinositol 3-kinase inhibitor, or hydroxy-2-naphthalenylmethylphosphonic acid, an insulin receptor inhibitor. While islets cultured in DAPT did not exhibit defects in insulin secretion, indicating that differentiation is unaltered, inhibiting gamma-secretase-dependent Notch activation led to a dose-dependent increase in caspase-3-dependent apoptosis in both MIN6 cells and human islets. Conversely, gamma-secretase overactivity resulted in an accumulation of cleaved NOTCH1 and protection from apoptosis.


CONCLUSIONS
Together these results show that the Notch/Ngn3 signalling network is intact and functional in adult islets. This pathway represents an attractive target for modulating beta cell fate in diabetes, islet transplantation and efforts to derive beta cell surrogates in vitro.
Dror
V
V
Laboratory of Molecular Signalling in Diabetes, Diabetes Research Group, Department of Cellular and Physiological Sciences and the Department of Surgery, University of British Columbia, Vancouver, BC, Canada.
Nguyen
V
V
Walia
P
P
Kalynyak
T B
TB
Hill
J A
JA
Johnson
J D
JD
D000368:Aged; D000375:Aging / Q000235:genetics / Q000502:physiology; D000818:Animals; D017209:Apoptosis / Q000235:genetics* / Q000502:physiology; D051017:Apoptosis Regulatory Proteins / Q000502:physiology; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000502:physiology; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D003470:Culture Media / Q000494:pharmacology; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008875:Middle Aged; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000502:physiology; D051881:Receptor, Notch1 / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology
D051017:Apoptosis Regulatory Proteins; D051792:Basic Helix-Loop-Helix Proteins; D053148:Caspase 3; D003470:Culture Media; D005947:Glucose; D036341:Intercellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D051881:Receptor, Notch1; C498695:NEUROG3 protein, human