Islet amyloid deposition and reduced ß-cell mass are pathologic hallmarks of type 2 diabetes. Amyloid deposits contain the 37 amino acid hIAPP, aggregation of which is toxic to ß cells. The 20-29 amino acid region of the peptide is known to confer its amyloidogenic potential. We previously reported that plasmin, a fibrinolytic protease, cleaves hIAPP thereby inhibiting fibril formation. We now sought to determine whether hIAPP fragments arising from plasmin-mediated cleavage retain the potential to aggregate and induce cytotoxicity. The ß-cell line INS-1 was treated for 24 h with full-length hIAPP (20 µM) alone or in the presence of plasmin (0.4 µM); plasmin alone or buffer were used as controls. Exposure to hIAPP alone decreased cell viability (53±1.4% of buffer, p<0.05, n=5). Addition of plasmin to hIAPP restored cell viability (90±4.3% of buffer, p=0.3 vs. buffer and p<0.05 vs. hIAPP alone, n=5), while treatment with plasmin alone had no effect (102±2.9% of buffer, p=0.7, n=5). LC/MS analysis of hIAPP cleavage products showed that plasmin induced a rapid decrease in the abundance of full-length hIAPP and appearance of the hIAPP 1-11 and 12-37 fragments. Since the hIAPP 12-37 contains the amyloidogenic region, we compared the amyloidogenic and cytotoxic properties of this fragment to full-length hIAPP. In a thioflavin T assay, hIAPP 12-37 was less amyloidogenic than full-length hIAPP. As expected, treatment of INS-1 cells for 24 h with full-length hIAPP (0-60 µM) reduced cell viability in a dose-dependent manner (67±1.3%, 30±0.4% and 16±0.7% of buffer, at 20, 40 and 60 µM respectively; p<0.05 vs. buffer for all, n=2), while hIAPP 12-37 was not toxic at any dose tested (90±3.2%, 90±3.3% and 108±3.5% of buffer, at 20, 40 and 60 µM respectively; n=2).
In summary, plasmin protects ß cells from hIAPP-induced toxicity by cleaving hIAPP between amino acids 11 and 12. Thus, increasing islet plasmin activity might be a strategy to limit ß-cell loss in type 2 diabetes.
N. Esser: None. M.F. Hogan: None. A.T. Templin: None. J.J. Castillo: None. D. Raleigh: None. J.S. Edgar: None. S. Zraika: Research Support; Self; Novartis Pharmaceuticals Corporation. R.L. Hull: None. S.E. Kahn: Advisory Panel; Self; Boehringer Ingelheim International GmbH, Eli Lilly and Company, Intarcia Therapeutics, Janssen Scientific Affairs, LLC., Merck & Co., Inc., Novo Nordisk A/S, Pfizer Inc.
U.S. Department of Veterans Affairs (I01BX001060)