Small interfering RNA-mediated suppression of proislet amyloid polypeptide expression inhibits islet amyloid formation and enhances survival of human islets in culture.
OBJECTIVE
Islet amyloid, formed by aggregation of the beta-cell peptide islet amyloid polypeptide (IAPP; amylin), is a pathological characteristic of pancreatic islets in type 2 diabetes. Toxic IAPP aggregates likely contribute to the progressive loss of beta-cells in this disease. We used cultured human islets as an ex vivo model of amyloid formation to investigate whether suppression of proIAPP expression would inhibit islet amyloid formation and enhance beta-cell survival and function.


METHODS
Islets from cadaveric organ donors were transduced with a recombinant adenovirus expressing a short interfering RNA (siRNA) designed to suppress human proIAPP (Ad-hProIAPP-siRNA), cultured for 10 days, and then assessed for the presence of islet amyloid, beta-cell apoptosis, and beta-cell function.


RESULTS
Thioflavine S-positive amyloid deposits were clearly present after 10 days of culture. Transduction with Ad-hProIAPP-siRNA reduced proIAPP expression by 75% compared with nontransduced islets as assessed by Western blot analysis of islet lysates 4 days after transduction. siRNA-mediated inhibition of IAPP expression decreased islet amyloid area by 63% compared with nontransduced cultured islets. Cell death assessed by transferase-mediated dUTP nick-end labeling staining was decreased by 50% in transduced cultured human islets, associated with a significant increase in islet insulin content (control, 100 +/- 4 vs. +Ad-siRNA, 153 +/- 22%, P < 0.01) and glucose-stimulated insulin secretion (control, 222 +/- 33 vs. +Ad-siRNA, 285 +/- 21 percent basal, P < 0.05).


CONCLUSIONS
These findings demonstrate that inhibition of IAPP synthesis prevents amyloid formation and beta-cell death in cultured human islets. Inhibitors of IAPP synthesis may have therapeutic value in type 2 diabetes.
Marzban
Lucy
L
Department of Pathology and Laboratory Medicine, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada. marzban@interchange.ubc.ca
Tomas
Alejandra
A
Becker
Thomas C
TC
Rosenberg
Lawrence
L
Oberholzer
Jose
J
Fraser
Paul E
PE
Halban
Philippe A
PA
Verchere
C Bruce
CB
D000256:Adenoviridae / Q000235:genetics; D000682:Amyloid / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D002470:Cell Survival / Q000235:genetics / Q000502:physiology; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000378:metabolism; D007515:Islets of Langerhans / Q000378:metabolism*; D009924:Organ Culture Techniques / Q000379:methods; D034741:RNA, Small Interfering / Q000235:genetics*; D014161:Transduction, Genetic
D000682:Amyloid; D034741:RNA, Small Interfering; C422904:pro-islet amyloid polypeptide