Islet amyloid in type 2 (non-insulin-dependent) diabetes is related to insulin

Diabetologia, May 1983

P. Westermark, E. Wilander

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Islet amyloid in type 2 (non-insulin-dependent) diabetes is related to insulin

Diabetologia Islet Amyloid in Type 2 (Non-Insulin-Dependent) Diabetes is Related to Insulin P. Westermark 0 1 E. Wilander 0 1 0 Department of Pathology, University of Uppsala , Uppsala , Sweden 1 Dr. P. Westermark Department of Pathology University Hospital S-751 85 Uppsala Sweden Summary. Amyloid deposition is the most typical islet alteration in Type 2 (non-insulin-dependent) diabetes. In the present study we show by immunohistochemistry that the amyloid reacts with an antiserum against insulin B chain. Islet amyloid was also purified, dissolved in guanidine-HC1 and gel filtered on a Sepharose 6B column. Immunization of a guinea pig with a high molecular weight fraction from this gel filtration resulted in an antiserum with insulin-binding capacity. This binding was partially blocked with pure insulin B chain. The results indicate that islet amyloid contains insulin B chain and that the amyloid is a product of the islet B cells. Thus the study support previous morphological studies. Insulin B chain; immunohistochemistry - 9 Springer-Verlag 1983 Amyloid deposits consist of fine fibrils, which are built up by low molecular weight proteins in fl-pleated sheet conformation [I]. Most important properties of the amyloid, such as resistance to enzymes and green birefringence after staining with Congo red, are explained by this conformation. Several different proteins have been shown to contribute to amyloid fibrils. These are: (a) homogeneous immunoglobulin light chains a n d / o r their variable segments in primary and myeloma associated amyloidosis [ 2 ], Co) protein AA in reactive (secondary) systemic amyloidosis [31, (c) pre-albumin or a pre-albumin-related protein in several forms of hereditary systemic amyloidosis and in senile cardiac amyloidosis [ 4, 5 ], and (d) a calcitonin-related protein in the amyloid of medullary carcinoma of the thyroid [6]. Hyalinization is the most typical alteration of the pancreatic islets in old people with Type 2 (non-insulindependent) diabetes mellitus [7-101. In this condition a hyaline material is deposited interstitially in the islets and is accompanied by a gradual loss of islet cells, especially the B cells [ 11, 12 ]. It has long been agreed that this material is a form of amyloid [ 8, 10, 13 ]. It has been suspected that the amyloid fibrils in the pancreatic islets and in insulinomas are formed by insulin [1], C-peptide [ 13 ] or some part of the preproinsulin molecule [ 14 ]. In the present paper, we show, using an immunoperoxidase method, that antiserum against insulin B chain binds to islet amyloid, while antiserum against A chain or insulin does not. We show, futhermore, that immunization of a guinea pig with a high molecular weight fraction of a partially purified islet amyloid resulted in an insulin-binding antiserum. Material and Methods Pancreatic tissue from patients with Type 2 diabetes was obtained at autopsies performed within 12 h after death. Pieces from the tail of the pancreas were fixed in Bouin's solution and embedded in paraffin. For examination of amyloid, sections were stained with Congo red and viewed in polarized light. The rest of the tail and the body of the pancreas was stored at - 20 ~ until used for purification of amyloid. Pancreatic tissue from six patients with heavy islet amyloidosis was used in the study (Table 1). 82 91 79 t0 16 ? Recently discovered 7 Treatment Chlorpropamide + metformin Chlorpropamide + metformin Diet ? None Unknown oral antidiabetic drug 40 ~o FRACTIONNUMBER 80 . . _ 4 , ~ 0.4 z< 0.3 An amyloid-rich material, which contained at least 25% of amyloid, was obtained from pancreases containing islets filled with amyloid, using a slight modification of the method by Pras et al. [ 15 ]. Briefly, the pancreatic tissue was homogenized repeatedly in normal saline until all soluble proteins had disappeared. The residue was in some cases digested with pepsin [ 16 ]. The residual insoluble material, which contained amyloid and some other tissue components, was lyophilized and then treated with guanidine HC1 (6 mol/1) in 0.1 mol/1 TrisHC1 buffer (pH 8.0) containing 0.2% EDTA and 0.1 mol/l dithiothreitol. After centrifugation (21,000 xg for 1 h), the dissolved material was gel-filtered on a Sepharose 6 B column equilibrated with guanidine HC1 (5 tool/l) in distilled water and eluted with the same solution under continuous registration of the absorbancy at 280 nm. Fractions were pooled, dialysed exhaustively against distilled water and lyophilized. Antisera Guinea pigs were immunized by intradermal injections bimonthly of the antigens mixed with Freund's complete adjuvant. The animals were bled by heart puncture 2 weeks after the third injection. Bovine insulin obtained from AB Vitrum, Stockholm, Sweden, oxidied bovine insulin A chain from Sigma Chemicals, Saint Louis, Missouri, USA and oxidied bovine insulin B chain from Boehringer, Mannheim, FGR were used as antigens. The purity of the insulinA an (...truncated)


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P. Westermark, E. Wilander. Islet amyloid in type 2 (non-insulin-dependent) diabetes is related to insulin, Diabetologia, 1983, pp. 342-346, Volume 24, Issue 5, DOI: 10.1007/BF00251821