A tripartite anoikis-like mechanism causes early isolated islet apoptosis.
BACKGROUND
This study examines the mechanisms of early isolated islet apoptosis (II-APO) and loss of functional islet mass.


METHODS
Rhesus islets were isolated for transplantation, and an aliquot was used for in vitro molecular studies of II-APO. These studies used Western blotting to examine caspase activation and perinuclear envelope protein cleavage that are associated with II-APO and used immunofluorescence analysis of Annexin V and mitochondrial permeability index to examine spontaneous and tripartite anoikis-like (TRAIL) mechanism--induced II-APO.


RESULTS
Caspase 6 was prominently activated in association with spontaneous II-APO, which occurred after overnight culture. In contrast, caspase 7, 8, and 9 were not activated. Cleavage of focal adhesion kinase and Lamin, substrates of caspase 6, was also evident in spontaneous II-APO. II-APO was exaggerated by the addition of the TRAIL mechanism. The TRAIL mechanism--induced II-APO was blocked by the caspase 6 inhibitor, VEID, and by the soluble fusion proteins, DR4 or DR5, which act as decoy receptors. In vivo studies in diabetic severe combined immunodeficiency disease mice showed that rhesus islets were cytoprotected by either ex vivo gene transfer of Bcl-2 or treatment of the isolated islet with VEID.


CONCLUSIONS
These studies suggest 3 major mechanisms involved in II-APO: caspase 6 activation, a TRAIL-induced apoptosis pathway, and the mitochondrial-associated apoptosis pathway. Inhibition of these II-APO pathways may improve isolated islet survival and reduce functional islet mass loss, which compromises the stable reversal of diabetes.
Thomas
F
F
Division of Transplantation, Department of Surgery, University of Alabama Medical Center, Birmingham, AL 35294-0012, USA.
Wu
J
J
Contreras
J L
JL
Smyth
C
C
Bilbao
G
G
He
J
J
Thomas
J
J
D000818:Animals; D023102:Anoikis / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D053178:Caspase 6; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D020287:In Situ Nick-End Labeling; D007515:Islets of Langerhans / Q000166:cytology* / Q000201:enzymology; D016381:Islets of Langerhans Transplantation; D008253:Macaca mulatta; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D016513:Mice, SCID; D008928:Mitochondria / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D053221:TNF-Related Apoptosis-Inducing Ligand; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D051017:Apoptosis Regulatory Proteins; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D008562:Membrane Glycoproteins; D009842:Oligopeptides; D053221:TNF-Related Apoptosis-Inducing Ligand; C505654:Tnfsf10 protein, mouse; D014409:Tumor Necrosis Factor-alpha; C110923:acetyl-valyl-isoleucyl-aspartyl-aldehyde; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse; C505516:Casp6 protein, mouse; D053178:Caspase 6; D020169:Caspases