Aberrant Processing of Human Proislet Amyloid Polypeptide Results in Increased Amyloid Formation

Diabetes, Jul 2005

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 NH2-terminal 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 β-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 β-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 β-cells.

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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 - 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)


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Johan F. Paulsson, Gunilla T. Westermark. Aberrant Processing of Human Proislet Amyloid Polypeptide Results in Increased Amyloid Formation, Diabetes, 2005, pp. 2117-2125, 54/7, DOI: 10.2337/diabetes.54.7.2117