Original article
Amyloid oligomers in diabetic and nondiabetic human pancreas

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The amyloid hypothesis of type 2 diabetes mellitus postulates that elevated levels of normally expressed monomeric proteins of human islet amyloid polypeptide (hIAPP) trigger oligomerization that independently causes fibril formation and disease progression. The aim of this study was to demonstrate the existence of amyloid oligomers in human pancreatic islets. Human pancreas tissues were obtained at autopsy of 8 nondiabetic control subjects (mean age = 75.8 ± 11.7 years, 4 males), 8 type 2 diabetic cases without islet amyloid (mean age = 78.8 ± 8.5 years, 4 males), and 8 type 2 diabetic patients with islet amyloid (mean age = 73.7 ± 14.2 years, 4 males). Several markers for insulin, IAPP, amyloid fibrils (thioflavin T), and apoptosis (cleaved caspase-3) were used in combination with an oligomer-specific antibody. Two distinct forms of oligomers were found in pancreatic islets. Small spherical puncta were found in approximately 3% to 20% of the islet cells of nondiabetic subjects, and large curvilinear structures as extracellular oligomers were identified frequently in diabetic islets. Large oligomers were spatially localized adjacent to amyloid fibrils and were associated with apoptosis. This report demonstrates the presence of 2 morphologic classes of amyloid oligomers in human pancreatic islets. The observations warrant function studies to investigate the clinical implications of the amyloid oligomerization in the pathogenesis of type 2 diabetes.

Section snippets

Experimental reagents

Full-length hIAPP1-37 peptides were custom synthesized at the Keck Biotechnology Center at Yale University and were purified in-house by reverse-phase high-performance liquid chromatography. The polyclonal rabbit antiamyloid oligomer antibody (Catalog# AB9234, A11) was purchased from Chemicon International (Temecula, Calif). This antibody binds specifically to cytotoxic oligomers formed by hIAPP, Alzheimer's Aβ, or other amyloidogenic peptides, but not to their monomeric or fibrillar form.3 The

Amyloid oligomers in human pancreatic islets

The specificity of the antioligomer antibody (A11) was shown by dot blot (Fig 1, A), whereas no crossreactivity between the antioligomer and anti-IAPP antibodies was detected by Western blot (Fig 1, B). The conformation-dependent structure of hIAPP-derived oligomers might be denatured by the SDS gel, and thus no oligomer-specific band was demonstrated by the Western blot assays. The Western blot assays with the anti-IAPP antibody showed hIAPP protein bands at the positions of 4 kD (monomers),

Discussion

This study provides direct evidence that (1) IAPP oligomers exist in adult human pancreatic islets; (2) hIAPP oligomers are either intracellular small puncta predominantly found in islet cells that express insulin/IAPP or are extracellular large curvilinear structures adjacent to fibril deposits; (3) oligomers of small spherical puncta are mainly found in nondiabetic islets, whereas larger curvilinear structures are predominantly detected in diabetic islets that include those islets with

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    Supported by grants CUHK4462/06M and CUHK4302/04M from the Research Grants Council of the Hong Kong Special Administrative Region, China.

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