A novel assay in vitro of human islet amyloid polypeptide amyloidogenesis and effects of insulin secretory vesicle peptides on amyloid formation.
Human islet amyloid polypeptide (IAPP) is a 37-residue peptide that is co-secreted with insulin by the beta-cell and might be involved in the pathogenesis of non-insulin-dependent diabetes mellitus. We developed an improved assay in vitro based on the fluorescence of bound thioflavin T to study factors affecting amyloidogenesis. Monomeric IAPP formed amyloid fibrils, as detected by increased fluorescence and by electron microscopy. Fluorimetric analysis revealed that the initial rate of amyloid formation was: (1) proportional to the peptide monomer concentration, (2) maximal at pH 9.5, (3) maximal at 200 mMKCl, and (4) proportional to temperature from 4 to 37 degreesC. We found that 5-fold and 10-fold molar excesses of proinsulin inhibited fibril formation by 39% and 59% respectively. Insulin was somewhat more potent with 5-fold and 10-fold molar excesses inhibiting fibril formation by 69% and 73% respectively, whereas C-peptide had no effect at these concentrations. Thus at physiological ratios of IAPP to insulin, insulin and proinsulin, but not C-peptide, can retard amyloidogenesis. Because insulin resistance or hyperglycaemia increase the IAPP-to-insulin ratio, increased intracellular IAPP compared with insulin expression in genetically predisposed individuals might contribute to intracellular amyloid formation, beta-cell death and the genesis of non-insulin-dependent diabetes mellitus.
Kudva
Y C
YC
Department of Medicine, Division of Endocrinology, Mayo Clinic, Rochester, MN 55906, USA.
Mueske
C
C
Butler
P C
PC
Eberhardt
N L
NL
D000682:Amyloid / Q000737:chemistry* / Q000648:ultrastructure; D052160:Benzothiazoles; D002096:C-Peptide / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000737:chemistry*; D003924:Diabetes Mellitus, Type 2 / Q000503:physiopathology; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000494:pharmacology; D007515:Islets of Langerhans / Q000737:chemistry*; D007700:Kinetics; D008854:Microscopy, Electron; D010446:Peptide Fragments / Q000737:chemistry* / Q000648:ultrastructure; D011384:Proinsulin / Q000494:pharmacology; D013696:Temperature; D013844:Thiazoles / Q000378:metabolism
D000682:Amyloid; D052160:Benzothiazoles; D002096:C-Peptide; D005456:Fluorescent Dyes; D007328:Insulin; D010446:Peptide Fragments; D013844:Thiazoles; C079438:islet amyloid polypeptide (8-37); C009462:thioflavin T; D011384:Proinsulin