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

Abstract

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 NH 2 -and COOH-terminal flanking peptides. This step is performed by the prohormone convertases PC2 and PC1/3. PC2 processes proIAPP preferably at the NH 2 -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 ␤-TC-6 cells expressing both prohormone convertases and in which proIAPP is processed into IAPP. Additionally P ancreatic islet amyloid is a frequent and characteristic pathological feature of type 2 diabetes. The amount of amyloid deposited varies, but it is present to some degree in 40 -95% of the patients (1-3), and a decrease in the number of ␤-cells follows the accumulation of islet amyloid (4 -6). The main amyloid constituent is the 37-amino 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 67-amino acid proIAPP molecule from which NH 2 -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 NH 2 -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 NH 2 -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 (17-19). 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 ...

The glucagon-like peptide-1 receptor agonist exenatide restores impaired pro-islet amyloid polypeptide processing in cultured human islets: implications in type 2 diabetes and islet transplantation

“…Enhanced prohIAPP processing in exenatide-treated human islets is associated with reduced amyloid formation during culture Impaired prohIAPP processing at its NH 2 -terminus has been shown to contribute to amyloid formation [30,31]. Therefore, we examined whether improved prohIAPP processing in exenatide-treated human islets can decrease formation of hIAPP aggregates, thereby reducing amyloid beta cell toxicity.…”

“…However, our studies suggest that exenatidetreated human islets release mainly mature hIAPP rather than immature (pro)hIAPP. Accordingly, previous studies have shown that an elevated hIAPP level per se, such as its overexpression in hIAPP-expressing mice, is not sufficient for amyloid formation [46] whereas elevated production and release of immature (pro)hIAPP potentiates hIAPP aggregation [30,31]. Importantly, improved prohIAPP processing by enhancing beta cell function in human islets with exenatide was associated with a lower number of islets containing hIAPP oligomers and amyloid deposits as well as a lower islet amyloid area in relation to total islet area compared with non-treated cultured islets.…”

“…Therefore, the latter step might be impaired due to insufficient processing time in the conditions associated with release of immature granules by beta cells. The impaired processing of prohIAPP, particularly at its NH 2 -terminal cleavage site, potentiates hIAPP aggregation thereby increasing amyloid-induced beta cell death [30,31]. Thus, restoring impaired prohIAPP processing may provide a new strategy to reduce hIAPP aggregation and its beta cell toxicity in conditions associated with islet amyloid formation such as type 2 diabetes, islet culture and transplantation.…”

“…Three factors have been proposed to contribute to amyloid formation in type 2 diabetes: (1) presence of an amyloidogenic sequence in the hIAPP molecule [26]; (2) elevated hIAPP production and secretion from beta cells associated with an increased demand for insulin [27][28][29] and (3) impaired prohIAPP processing associated with beta cell dysfunction [28,30,31]. The mechanisms underlying amyloid formation in cultured and transplanted human islets are still unclear but lower clearance of hIAPP secreted from beta cells due to the disrupted vasculature and beta cell dysfunction caused by islet culture and transplantation may contribute to this process.…”

Loss of prohormone convertase 2 promotes beta cell dysfunction in a rodent transplant model expressing human pro-islet amyloid polypeptide

“…Overexpression of human proIAPP in PC1/3-and PC2-deficient cell lines promotes increased amyloid deposition and apoptosis [28]. We have previously demonstrated that cultured human proIAPP-overexpressing islets lacking PC2 develop increased amyloid formation and cell apoptosis compared with islets with normal PC2 levels [29].…”