Aberrant Processing of Human Proislet Amyloid Polypeptide Results in Increased Amyloid Formation
Johan F. Paulsson
0
1
Gunilla T. Westermark
0
1
0
Diabetes Research Center, Linko ping University
, Linko ping,
Sweden
. PhD,
Cell Biology, Linko ping University
, SE 581 85 Linko ping,
Sweden
1
Department of Biomedicine and Surgery, Division of Cell Biology, Linko ping University
, Linko ping,
Sweden
; and the
The amyloid present in the islets of Langerhans in type 2 diabetes is polymerized islet amyloid polypeptide (IAPP). The precursor protein proIAPP is posttranslationally modified, a process involving the removal of NH2- and COOH-terminal flanking peptides. This step is performed by the prohormone convertases PC2 and PC1/3. PC2 processes proIAPP preferably at the NH2terminal processing site, and PC1/3 processes proIAPP exclusively at the COOH-terminal site. Little is known regarding the exact circumstances leading to islet amyloid formation. In this study, we have examined the possible significance of aberrant processing of proIAPP on amyloid formation in several in vitro cellular systems. In our studies, human (h)-proIAPP was transfected into -TC-6 cells expressing both prohormone convertases and in which proIAPP is processed into IAPP. Additionally, h-proIAPP was transfected into three different pituitary-derived cell lines with different prohormone convertase profiles: AtT-20 cells (deficient in PC2), GH3 cells (deficient in PC1/3), and GH4C1 cells (deficient in both convertases). We followed the processing of h-proIAPP with antibodies specific for the respective cleavage sites and stained the cells with Congo red to verify the accumulation of amyloid. Incomplete processing of h-proIAPP that occurs in AtT-20 and GH4C1 cells resulted in the formation of intracellular amyloid. No amyloid developed in -TC-6 and GH3 cells lines with full processing of proIAPP. An intracellular increase in proIAPP and/or its metabolic products may thus promote intracellular amyloid formation, thereby causing cell death. When extracellularly exposed, this amyloid might act as template for continuing amyloid formation from processed IAPP released from the surrounding -cells. Diabetes 54:2117-2125, 2005
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PThe amount of amyloid deposited varies, but it is
ancreatic islet amyloid is a frequent and
characteristic pathological feature of type 2 diabetes.
present to some degree in 40 95% of the patients
(13), and a decrease in the number of -cells follows the
accumulation of islet amyloid (4 6). The main amyloid
constituent is the 37amino acid polypeptide hormone
islet amyloid polypeptide (IAPP) (7) or amylin (8). This
-cell product is stored (9) and released together with
insulin upon stimulation (10,11). In humans, IAPP is
synthesized as a 67amino acid proIAPP molecule from
which NH2-terminal and COOH-terminal flanking peptides
are subsequently removed proteolytically (12). This
posttranslational processing within the secretory granules
occurs at di-basic amino acids and is performed by the
prohormone convertases 2 and 1/3 (PC2 and PC1/3). These
are the same enzymes that process proinsulin to insulin at
the same cellular location (13). In in vitro studies on the
processing of human proIAPP (h-proIAPP) by
recombinant converting enzymes, PC2 favored processing at the
NH2-terminal flanking region and PC1/3 had its initial
activity at the COOH-terminal region. However, during
extended incubation time, both PC2 and PC1/3 could, to
some degree, process proIAPP at both cleavage sites (14).
In contrast, the in vivo results from studies on the
processing of proIAPP performed in PC2 (15) and PC1/3 (16)
null mice showed that PC2 is required for cleavage at the
NH2-terminal region and that PC1/3 processes the
COOHterminal flanking region. However, in the absence of
PC1/3, this cleavage site can be processed by PC2.
Little is known about the mechanisms that precede the
deposition of islet amyloid. The use of human IAPP
(h-IAPP) expressing transgenic animals has
demonstrated that increased expression of IAPP by itself is not
sufficient for amyloid to develop (1719). However,
introduction of the h-IAPP gene into mouse strains with
diabetic traits resulted in the formation of islet amyloid
(20,21). Amyloid was also detected in transgenic animals
fed a high-fat diet (22). In mouse IAPP (mIAPP)null mice
expressing the gene for h-IAPP, amyloid developed within
9 months when fed a high-fat diet (23).
Human islet amyloid observed postmortem is almost
exclusively extracellular. Early amyloidogenesis has thus
been thought to occur outside the -cells (24). However,
studies in transgenic mice showed that early amyloid also
appeared intracellularly in some cells in affected islets.
Intracellular amyloid has also been observed in human
islet grafts implanted subcapsularly in the kidneys of nude
mice. In this mouse model for islet transplantation,
intracellular IAPP amyloid was present in 75% of the implants
within 2 weeks after implantation (25). In humans,
intracellular amyloid has also been observed in insulinomas
(26). These findings may in (...truncated)