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)