A novel assay in vitro of human islet amyloid polypeptide amyloidogenesis and effects of insulin secretory vesicle peptides on amyloid formation

Yogish C. Kudva, Cheryl Mueske, Peter C. Butler, Norman L. Eberhardt

Research output: Contribution to journalArticle

55 Scopus citations

Abstract

Human islet amyloid polypeptide (IAPP) is a 37-residue peptide that is co-secreted with insulin by the β-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 mM KCl, and (4) proportional to temperature from 4 to 37°C. 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, β-cell death and the genesis of non-insulin-dependent diabetes mellitus.

Original languageEnglish (US)
Pages (from-to)809-813
Number of pages5
JournalBiochemical Journal
Volume331
Issue number3
DOIs
StatePublished - May 1 1998

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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