3T3-L1 adipocytes were incubated in serum-free media at 37°C with (dashed line) or without (solid line) 100 ng͞ml insulin for 8 h. Isoproterenol (10 M) was then added for the indicated time course at 37°C, and intracellular cAMP was determined by enzyme immunoassay, as described in Materials and Methods. Data are from a typical experiment done in triplicate wells Ϯ SEM and are representative of three separate experiments. (B) 3T3-L1 adipocytes were incubated in serum-free media for 8 h. Where indicated, 100 ng͞ml insulin was added for 15 min before isoproterenol treatment. Cells were then treated with 10 M isoproterenol for 5 min at 37°C, and intracellular cAMP was determined by enzyme immunoassay, as described in Materials and Methods. Data are from a typical experiment done in triplicate wells Ϯ SEM and are representative of three separate experiments.www.pnas.org͞cgi͞doi͞10.1073͞pnas.0630528100 After this, cells were treated with 10 M isoproterenol for 5 min at 37°C. Cells were then washed with PBS at 37°C and restimulated with a second 10 M dose of isoproterenol for an additional 5 min at 37°C; intracellular cAMP was determined by enzyme immunoassay. (B) 3T3-L1 adipocytes were incubated at 37°C in serum-free media for 8 h in the presence (dashed lines) or absence (solid lines) of 100 ng͞ml insulin. Forskolin was added for the final 5 min, and intracellular cAMP levels were determined by enzyme-linked immunoassay. Data are from typical experiments done in triplicate wells and are representative of three separate experiments.

Secondary, or amyloid protein A (AA), amyloidosis is a complication of chronic inflammatory diseases, both infectious and noninfectious. AA constitutes the insoluble fibrils, which are deposited in different organs, and is a major N-terminal part of the acute phase protein serum AA. It is not known why only some patients with chronic inflammation develop AA amyloidosis. Nucleation is a widely accepted mechanism in amyloidogenesis. Preformed amyloid-like fibrils act as nuclei in amyloid fibril formation in vitro, and AA amyloid fibrils and synthetic amyloid-like fibrils also may serve as seed for fibril formation in vivo. In addition to amyloid fibrils, there is a variety of similar nonmammalian protein fibrils with ␤-pleated structure in nature. We studied three such naturally occurring protein fibrils: silk from Bombyx mori, Sup35 from Saccharomyces cerevisiae, and curli from Escherichia coli. Our results show that these protein fibrils exert amyloid-accelerating properties in the murine experimental AA amyloidosis, suggesting that such environment factors may be important risk factors in amyloidogenesis.amyloid ͉ seeding ͉ protein misfolding ͉ aggregation ͉ prion