| The pathology of type II diabetes includes the presence of cytotoxic amyloid deposits in the islets of Langerhans, The main component of these deposits, islet amyloid polypeptide (IAPP), is a hormone involved in glucose metabolism and is normally co-secreted with insulin by the β-cells of the pancreas. Although the role of islet amyloid in the pathogenesis of the disease is unclear, amyloid is localized in vivo with areas of cell degeneration and amyloid fibers produced in vitro are toxic to cultured cells.; The mechanism by which soluble IAPP converts into insoluble aggregates has not been well established. In order to better understand this process, a novel method for monitoring fibrillogenesis has been developed. The approach, based on electrospray ionization mass spectrometry, is complementary to existing assays of fiber formation as it monitors directly the consumption of soluble IAPP rather than the formation of end product. We use this method to monitor the consumption of soluble IAPP in seeded reactions as well as measure the equilibrium monomer concentration after the reaction is complete.; Changes in the concentration of IAPP relative to other granule components may be responsible for amyloid formation in type II diabetes. To investigate the mechanism by which insulin acts on fiber formation, we perform in vitro IAPP fibrillogenesis experiments in the presence and absence of insulin. We find that insulin is an exceptionally potent inhibitor. IAPP fiber formation in vitro requires a minimum of three steps: fiber-independent nucleation, elongation, and fiber-dependent nucleation. Furthermore, these steps are attenuated by the presence of a dispersed-phase transition. We interpret this data in the context of the phase-mediated fibrillogenesis model (PMF) and conclude through experiment and kinetic simulation that the dominant effect of insulin is to act on the elongation portion of the reaction. Unusually, the magnitude of the inhibitory effect is dependent on insulin concentration, yet independent of IAPP concentration. This indicates that a disruption in insulin:IAPP ratios is unlikely to be the primary cause of amyloid formation. These results further suggest that amyloid formation in type II diabetes involves either an additional agent that acts as an accelerant, or a step that segregates IAPP from insulin. |
