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Islet amyloid polypeptide (IAPP): Mechanisms of Amyloidogenesis in the Pancreatic Islets and Potential Roles in Diabetes Mellitus
Linköping University, Department of Molecular and Clinical Medicine, Molecular and Immunological Pathology. Linköping University, Faculty of Health Sciences.
2001 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Islet amyloid is the most common characteristic feature of the islets in type 2 diabetes, being found in up to 90% of diabetic patients at post-mortem. lt has as its unique component the islet beta-cell peptide islet amyloid polypeptide (IAPP), which is eo-secreted with insulin. Because all human subjects produce and secrete the amyloidogenic form of IAPP, yet not all develop islet amyloid, some other factors must be involved in islet amyloid formation. The aim of the research presented in this thesis was to study factors of importance for the IAPP amyloidogenesis in type 2 diabetes. We developed a mouse monoclonal antibody to raVmouse IAPP (MAb4A5). MAb4A5 shows reactivity with IAPP in different species without detecting its close relative CGRP. In the pancreatic islets from patients with type 2 diabetes and diabetic cat, MAb4A5 labels immunohistochemically cellular IAPP but not IAPP in islet amyloid deposits. In contrast to MAb4A5 polyclonal rabbitiAPP antisera label beta cells close to amyloid only weakly, but label strongly IAPP in its amyloid form. The varying findings of IAPP immunoreactivity in pancreatic islets indicate that IAPP undergoes structural changes (impaired cleavage of proiAPP, conformational change, or post-transitional modifications) during the amyloidogenesis. A potentially important finding was the increased IAPP immunoreactivity in beta cells in islets of impaired glucose tolerant cats, irrespective of presence of amyloid in these islets. The finding may indicate that the formation of first islet amyloid occurs before Type 2 diabetes is manifest. Given the immunohistochemical results with MAb 4A5, we investigated whether the altered immunoreactivity of IAPP in association with the amyloidogenesis is due to a modification of IAPP (e.g. non-enzymatic glycation). We used synthetic IAPP fibrils glycated in vitro to study if non-enzymatic glycation may result in loss of an antigenic epitope. The results showed that a possible explanation of the lack of immunoreactivity of islet amyloid with MAb 4A5 actually is an nonenzymatic glycation. Association of an IAPP gene mutation with Type 2 diabetes has been found in the Japanese and Chinese population. We studied the possible enhanced fibril formation capacity of the mutant IAPP in vitro. Full-length and truncated IAPPS20G can form more amyloid-like fibrils and do this faster compared to wild type IAPP in vitro. We concluded that mutant (S20G) IAPP is a more amyloidogenic IAPP molecule and may be associated with an increased islet amyloid formation in vivo.

Based on the reports on the occurrence of islet amyloid in transgenic mice fed high fat diet, we investigated effects of free fatty acids on IAPP amyloid formation in isolated islets from transgenic mice expressing the gene for human IAPP but deficient of endogenous murine IAPP, and effects of FFAs on polymerization of IAPP in vitro. We found free fatty acids accelerate and increase polymerization of IAPP in vitro and promote amyloid like aggregation occurring in cultivated transgenic mouse isolated islets. All these results indicate that the pathogenesis of the islet amyloid may be a complex process involving many different mechanisms.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2001. , 70 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 655
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-28607Local ID: 13762ISBN: 91-7219-756-0 (print)OAI: oai:DiVA.org:liu-28607DiVA: diva2:249418
Public defence
2001-01-12, Berzeliussalen, Universitetssjukhuset, Linköping, 13:30 (Swedish)
Opponent
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2012-08-17Bibliographically approved
List of papers
1. Altered immunoreactivity of islet amyloid polypeptide (IAPP) may reflect major modifications of the IAPP molecule in amyloidogenesis
Open this publication in new window or tab >>Altered immunoreactivity of islet amyloid polypeptide (IAPP) may reflect major modifications of the IAPP molecule in amyloidogenesis
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1997 (English)In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 40, no 7, 793-801 p.Article in journal (Refereed) Published
Abstract [en]

We have developed a mouse monoclonal antibody against rat/mouse islet amyloid polypeptide (IAPP). The antibody recognises an epitope in the N-terminal part of the molecule, which is conserved between different species. The antibody immunohistochemically labelled beta cells in normal islets of most different mammalian species including man and in one avian species. Previous immunohistochemical studies of human pancreatic tissue from individuals with non-insulin-dependent diabetes mellitus (NIDDM) have revealed a paradoxical and unexplained lack of IAPP immunoreactivity in beta cells close to amyloid in spite of the presence of IAPP mRNA. In contrast to these findings we show that the newly developed monoclonal IAPP antibody strongly labels such beta cells while islet amyloid deposits which are labelled by polyclonal antisera do not bind the monoclonal antibody. These findings with the polyclonal antisera and the monoclonal antibody indicate that IAPP undergoes one or several structural changes during the amyloidogenesis. Knowledge of these structural changes that may include abnormal folding or chemical modification of IAPP is probably important for the understanding of the amyloidogenesis and the pathogenesis of the islet lesion in NIDDM.

Keyword
islet amyloid polypeptide, monoclonal antibody, non-insulin-dependent diabetes mellitus, immunohistochemistry, deposits.
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-80001 (URN)10.1007/s001250050751 (DOI)
Available from: 2012-08-17 Created: 2012-08-17 Last updated: 2017-12-07Bibliographically approved
2. Quantitative immunohistochemical analysis of islet amyloid polypeptide (IAPP) in normal, impaired glucose tolerant, and diabetic cats
Open this publication in new window or tab >>Quantitative immunohistochemical analysis of islet amyloid polypeptide (IAPP) in normal, impaired glucose tolerant, and diabetic cats
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1998 (English)In: Amyloid: Journal of Protein Folding Disorders, ISSN 1350-6129, E-ISSN 1744-2818, Vol. 5, no 4, 255-261 p.Article in journal (Refereed) Published
Abstract [en]

Islet amyloid polypeptide (IAPP, “amylin”) has been proposed as having important roles in the pathogenesis of type 2 diabetes mellitus via its biological activity and by forming islet amyloid. The domestic cat develops a type of diabetes that closely resembles type 2 diabetes in humans, including the frequent formation of islet amyloid deposits in the impaired glucose tolerant (IGT) and diabetic state. With the aid of computerized image analysis and immuno-histochemistry, we examined the IAPP and insulin content inpancreatic islets of normal, IGT and diabetic cats. IAPP immunoreactivity in beta cells from IGT cats was significantly stronger (p < 0.01) as compared with cells from normal cats, while the insulin labelling strength was unchanged. Overtly diabetic cats were usually almost devoid of beta cells. As in humans, cellular IAPP but not IAPP in islet amyloid deposits was labelled by the newly developed monoclonal antibody to IAPP 4A5, thus providing further evidence that IAPP is modified by a yet unknown mechanism during the amyloidogenic process. The study provides evidence that an increased beta cell storage of IAPP independent of insulin may be an important factor in the early phase of the development of islet amyloid in this form of diabetes.

Keyword
islet amyloid polypeptide, impaired glucose tolerance, non-insulin-dependent diabetes mellitus, immunohistochemistry, cat, amyloidosis
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-80002 (URN)10.3109/13506129809007298 (DOI)
Available from: 2012-08-17 Created: 2012-08-17 Last updated: 2017-12-07Bibliographically approved
3. Amyloid in Human Islets of Langerhans: Immunologic Evidence That Islet Amyloid Polypeptide Is Modified in Amyloidogenesis
Open this publication in new window or tab >>Amyloid in Human Islets of Langerhans: Immunologic Evidence That Islet Amyloid Polypeptide Is Modified in Amyloidogenesis
2000 (English)In: Pancreas, ISSN 0885-3177, E-ISSN 1536-4828, Vol. 21, no 2, 212-218 p.Article in journal (Refereed) Published
Abstract [en]

Amyloid derived from the beta-cell product islet amyloid polypeptide (IAPP) has been implicated for a beta-cell lesion in Type II diabetes mellitus. The pathogenesis of islet amyloid is poorly understood, and in addition to an amyloidogenic IAPP molecule and possibly increased concentration of IAPP, other unknown factors seem to be included. It was shown previously that polyclonal rabbit IAPP antisera label beta cells close to amyloid only weakly. Whether this lack of immunoreactivity depends on lack of IAPP or on hidden epitopes is in question. In the present study, we show that the IAPP immunoreactivity of these beta cells is possible to retrieve. On the other hand, the monoclonal IAPP antibody 4A5, which labels IAPP in beta cells, does not label IAPP in its native amyloid form. We show evidence that this lack of immunoreactivity is not dependent on conformational change of the IAPP molecules in the amyloidogenesis but is likely owing to glycation of IAPP in human islet amyloid deposits.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25449 (URN)10.1097/00006676-200008000-00015 (DOI)9895 (Local ID)9895 (Archive number)9895 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
4. Enhanced in vitro production of amyloid-like fibrils from mutant (S20G) islet amyloid polypeptide
Open this publication in new window or tab >>Enhanced in vitro production of amyloid-like fibrils from mutant (S20G) islet amyloid polypeptide
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2001 (English)In: Amyloid: Journal of Protein Folding Disorders, ISSN 1350-6129, E-ISSN 1744-2818, Vol. 8, no 4, 242-249 p.Article in journal (Refereed) Published
Abstract [en]

Islet amyloid polypeptide (IAPP, “amylin”) is the amyloid-fibril-forming polypeptide in the islets of Langerhans associated with type 2 diabetes mellitus. A missense mutation in the IAPP gene associated with early-onset type 2 diabetes has been identified in the Japanese population. This mutation results in a glycine for serine substitution at position 20 of the mature IAPP molecule. Whether or not formation of islet amyloid with resulting destruction of islet tissue is the cause of this diabetes is yet not known. The present in vitro study was performed in order to investigate any influence of the amino acid substitution on the fibril formation capacity. Synthetic full-length wild type (lAPPwt) and mutant (IAPPS20G) as well as corresponding truncated peptides (position 18-29) were dissolved in dimethylsulfoxide (DMSO) or in 10% acetic acid at a concentration of 10 mg/mL and their fibril forming capacity was checked by Congo red staining, electron microscopy, a Congo red affinity assay and Thioflavine T fluorometric assay. It was found that full-length and truncated IAPPS20G both formed more amyloid-like fibrils and did this faster compared to IAPPwt. The fibril morphology differed slightly between the preparations. Conclusion: The amino acid substitution (S20G) is situated close to the region of the IAPP molecule implicated in the IAPP fibrillogenesis. The significantly increased formation of amyloid-like fibrils by IAPPS20G is highly interesting and may be associated with an increased islet amyloid formation in vivo and of fundamental importance in the pathogenesis of this specific form of diabetes.

Keyword
Islet amyloid polypeptide, fibrillogenesis, mutation, dye fluorescence, type 2 diabetes
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25451 (URN)10.3109/13506120108993820 (DOI)9897 (Local ID)9897 (Archive number)9897 (OAI)
Note
On the day of the defence day the status of this article was submittedAvailable from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
5. Effects of free fatty acid on polymerization of islet amyloid polypeptide (IAPP) in vitro and on amyloid fibril formation in cultivated isolated islets of transgenic mice overexpressing human IAPP
Open this publication in new window or tab >>Effects of free fatty acid on polymerization of islet amyloid polypeptide (IAPP) in vitro and on amyloid fibril formation in cultivated isolated islets of transgenic mice overexpressing human IAPP
2002 (English)In: Molecular medicine (Cambridge, Mass. Print), ISSN 1076-1551, E-ISSN 1528-3658, Vol. 8, no 12, 863-868 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Islet amyloid polypeptide (IAPP) is deposited as amyloid in the islets of Langerhans in type 2 diabetes. The mechanism behind the formation of the cytotoxic fibrils is unknown. Islet amyloid develops in a mouse IAPP null mouse strain that expresses human IAPP (+hIAPP/-mIAPP) after 9 months on a high-fat diet. Herein we investigate the effect that individual free fatty acids (FFAs) exert on formation of amyloid-like fibrils from synthetic IAPP and the effects of FFAs on IAPP polymerization in +hIAPP/-mIAPP islets cultivated in vitro.

MATERIALS AND METHODS: In the study myristic acid, palmitic acid, stearic acid, oleic acid, and linoleic acid were used together with albumin. Thioflavin T (Th T) assay was used for quantification of amyloid-like fibrils. Islets were isolated from the +hIAPP/-mIAPP transgenic strain and cultured in the presence of the FFAs for 2 days. Immuno-electron microscopy was used for evaluation.

RESULTS: The Th T assay showed that all studied FFAs potentiated fibril formation but that myristic acid revealed the highest capacity. In some cells from cultured islets, intragranular aggregates were present. These aggregates had a filamentous appearance and labeled with antibodies against IAPP. In some cells cultured in the presence of linoleic acid, large amounts of intracellular amyloid were present. Earlier, this has not been observed after such a short incubation period.

CONCLUSIONS: Our studies suggest that FFAs can potentiate amyloid formation in vitro, probably without being integrated in the fibril. Cultivation of +hIAPP/-mIAPP transgenic mouse islets with FFAs results in altered morphology of the secretory granules with appearance of IAPP- immunoreactive fibrillar material. We suggest that such fibrillar material may seed extracellular amyloid formation after exocytosis.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26538 (URN)11099 (Local ID)11099 (Archive number)11099 (OAI)
Note
On the day of the defence day the status of this article was a manuscriptAvailable from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved

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