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1.
Cell Death Dis ; 2: e232, 2011 Nov 24.
Article in English | MEDLINE | ID: mdl-22113197

ABSTRACT

Diabetes is associated with the death and dysfunction of insulin-producing pancreatic ß-cells. In other systems, Musashi genes regulate cell fate via Notch signaling, which we recently showed regulates ß-cell survival. Here we show for the first time that human and mouse adult islet cells express mRNA and protein of both Musashi isoforms, as well Numb/Notch/Hes/neurogenin-3 pathway components. Musashi expression was observed in insulin/glucagon double-positive cells during human fetal development and increased during directed differentiation of human embryonic stem cells (hESCs) to the pancreatic lineage. De-differentiation of ß-cells with activin A increased Msi1 expression. Endoplasmic reticulum (ER) stress increased Msi2 and Hes1, while it decreased Ins1 and Ins2 expression, revealing a molecular link between ER stress and ß-cell dedifferentiation in type 2 diabetes. These effects were independent of changes in Numb protein levels and Notch activation. Overexpression of MSI1 was sufficient to increase Hes1, stimulate proliferation, inhibit apoptosis and reduce insulin expression, whereas Msi1 knockdown had the converse effects on proliferation and insulin expression. Overexpression of MSI2 resulted in a decrease in MSI1 expression. Taken together, these results demonstrate overlapping, but distinct roles for Musashi-1 and Musashi-2 in the control of insulin expression and ß-cell proliferation. Our data also suggest that Musashi is a novel link between ER stress and the compensatory ß-cell proliferation and the loss of ß-cell gene expression seen in specific phases of the progression to type 2 diabetes.


Subject(s)
Apoptosis , Diabetes Mellitus, Type 2/metabolism , Endoplasmic Reticulum Stress/physiology , Insulin-Secreting Cells/metabolism , Insulin/metabolism , Nerve Tissue Proteins/metabolism , RNA-Binding Proteins/metabolism , Activins/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Differentiation , Cell Proliferation , Cells, Cultured , Diabetes Mellitus, Type 2/pathology , Embryonic Stem Cells/cytology , Gene Expression Regulation , Homeodomain Proteins/metabolism , Humans , Mice , Nerve Tissue Proteins/antagonists & inhibitors , RNA-Binding Proteins/antagonists & inhibitors , Receptors, Notch/metabolism , Transcription Factor HES-1
2.
Diabetologia ; 50(12): 2504-15, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17922104

ABSTRACT

AIMS/HYPOTHESIS: 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/INTERPRETATION: 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.


Subject(s)
Apoptosis/genetics , Islets of Langerhans/physiology , Receptor, Notch1/physiology , Aged , Aging/genetics , Aging/physiology , Animals , Apoptosis/physiology , Apoptosis Regulatory Proteins/physiology , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/physiology , Caspase 3/genetics , Caspase 3/metabolism , Cells, Cultured , Culture Media/pharmacology , Female , Gene Expression Regulation/physiology , Glucose/pharmacology , Humans , Intercellular Signaling Peptides and Proteins/pharmacology , Islets of Langerhans/drug effects , Islets of Langerhans/metabolism , Male , Mice , Mice, Inbred C57BL , Middle Aged , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/physiology , Receptor, Notch1/genetics , Signal Transduction/genetics , Signal Transduction/physiology
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