Amyloid Deposition in Transplanted Human Pancreatic Islets: A Conceivable Cause of Their Long-Term Failure

Following the encouraging report of the Edmonton group, there was a rejuvenation of the islet transplantation field. After that, more pessimistic views spread when long-term results of the clinical outcome were published. A progressive loss of the β-cell function meant that almost all patients were back on insulin therapy after 5 years. More than 10 years ago, we demonstrated that amyloid deposits rapidly formed in human islets and in mouse islets transgenic for human IAPP when grafted into nude mice. It is, therefore, conceivable to consider amyloid formation as one potential candidate for the long-term failure. The present paper reviews attempts in our laboratories to elucidate the dynamics of and mechanisms behind the formation of amyloid in transplanted islets with special emphasis on the impact of long-term hyperglycemia

Proislet Amyloid Polypeptide (proIAPP) : Impaired Processing is an Important Factor in Early Amyloidogenesis in Type 2 Diabetes

Amyloid is defined as extracellular protein aggregates with a characteristic fibrillar ultra-structure, Congo red affinity and a unique x-ray diffraction pattern. At present, 25 different human amyloid fibril proteins have been identified, and amyloid aggregation is associated with pathological manifestations such as Alzheimer’s disease, spongiform encephalopathy and type 2 diabetes. Amyloid aggregation triggers apoptosis by incorporation of early oligomers in cellular membranes, causing influx of ions. Amyloid is the only visible pathological islet alteration in subjects with type 2 diabetes, and islet amyloid polypeptide (IAPP) is the major islet amyloid fibril component. IAPP is produced by beta-cells and co-localized with insulin in the secretory granules. Both peptides are synthesised as pro-molecules and undergo proteolytic cleavage by the prohormone convertase 1/3 and 2. Although IAPP is the main amyloid constituent, both proIAPP and proIAPP processing intermediates have been identified in islet amyloid. The aim of this thesis was to study the role of impaired processing of human proIAPP in early islet amyloidogenesis. Five cell lines with individual processing properties were transfected with human proIAPP and expression, aggregation and viability were studied. Cells unable to process proIAPP into IAPP or to process proIAPP at the N-terminal processing site accumulated intracellular amyloid-like aggregates and underwent apoptosis. Further, proIAPP immunoreactivity was detected in intracellular amyloid-like aggregates in betacells from transgenic mice expressing human IAPP and in transplanted human beta-cells. ProIAPP was hypothesized to act as a nidus for further islet amyloid deposition, and to investigate this theory, amyloid-like fibrils produced from recombinant IAPP, proIAPP and insulin C-peptide/A-chain were injected in the tail vein of transgenic mice expressing the gene for human IAPP. Pancreata were recovered after 10 months and analysed for the presence of amyloid. Both IAPP and proIAPP fibrils but not des-31,32 proinsulin fibrils, caused an increase in affected islets and also an increase of the amyloid amount. This finding demonstrates a seeding capacity of proIAPP on IAPP fibrillogenesis. IAPP has been known for some time to trigger apoptosis in cultured cells, and a novel method for real time detection of apoptosis in beta-cells was developed. Aggregation of recombinant proIAPP and proIAPP processing intermediates were concluded to be inducers of apoptosis as potent as IAPP fibril formation. From the results of this study, a scenario for initial islet amyloidogenesis is proposed. Initial amyloid formation occurs intracellularly as a result of alterations in beta-cell processing capacity. When the host cell undergoes apoptosis intracellular proIAPP amyloid becomes extracellular and can act as seed for further islet amyloid deposition

Real-Time Monitoring of Apoptosis by Caspase-3-Like Protease Induced FRET Reduction Triggered by Amyloid Aggregation

Amyloid formation is cytotoxic and can activate the caspase cascade. Here, we monitor caspase-3-like activity as reduction of fluorescence resonance energy transfer (FRET) using the contstruct pFRET2-DEVD containing enhanced cyan fluorescent protin (EYFP) linked by the caspase-3 specific cleavage site residues DEVD. Beta-TC-6 cells were transfected, and the fluoorescence was measured at 440 nm excitation and 535 nm (EYFP) and 480 nm (ECFP) emission wavelength. Cells were incubated with recombinant pro lset Amyloid Polypeptide (rec prolAPP) or the processing metabolites of prolAPP; the N-terminal flanking peptide withIAPP (recN+IAPP); IAPP with the C-terminal flanking peptied (recIAPP+C) and lslet Amyloid Polypeptide (recIAPP). Peptides were added in solubilized from (50 mu M) or as performed amyloid-like fibrils, or as a combination of these. FRET was measured and incubation with a mixture of solubilized peptide and performed fibrils resulted in loss of FRET and apoptosis was determined to occurein cells incubated with recproIAPP (49%), recN+IAPP (46%), recIAPP (72%) and recIAPP+C (59%). These results show that proIAPP and the processing intermediates reside the same cell toxic capacity as IAPP, and they can all have a central role in the reduction of beta-cell number in type 2 diabetes.Original Publication:Johan F Paulsson, Sebastian Schultz, Martin Kohler, Ingo Leibiger, Per-Olof Berggren and Gunilla Westermark, Real-Time Monitoring of Apoptosis by Caspase-3-Like Protease Induced FRET Reduction Triggered by Amyloid Aggregation, 2008, EXPERIMENTAL DIABETES RESEARCH, (2008), 865850.http://dx.doi.org/10.1155/2008/865850Copyright: Author

Elucidation of the Binding Mode of the Carboxyterminal Region of Peptide YY to the Human Y-2 Receptor

Understanding the agonist-receptor interactions in the neuropeptide Y (NPY)/peptide YY (PYY) signaling system is fundamental for the design of novel modulators of appetite regulation. We report here the results of a multidisciplinary approach to elucidate the binding mode of the native peptide agonist PYY to the human Y2 receptor, based on computational modeling, peptide chemistry and in vitro pharmacological analyses. The preserved binding orientation proposed for full-length PYY and five analogs, truncated at the amino terminus, explains our pharmacological results where truncations of the N-terminal proline helix showed little effect on peptide affinity. This was followed by receptor mutagenesis to investigate the roles of several receptor positions suggested by the modeling. As a complement, PYY-(3-36) analogs were synthesized with modifications at different positions in the common PYY/NPY C-terminal fragment (32TRQRY36-amide). The results were assessed and interpreted by molecular dynamics and Free Energy Perturbation (FEP) simulations of selected mutants, providing a detailed map of the interactions of the PYY/NPY C-terminal fragment with the transmembrane cavity of the Y2 receptor. The amidated C-terminus would be stabilized by polar interactions with Gln2886.55 and Tyr2195.39, while Gln1303.32 contributes to interactions with Q34 in the peptide and T32 is close to the tip of TM7 in the receptor. This leaves the core, α-helix of the peptide exposed to make potential interactions with the extracellular loops. This model agrees with most experimental data available for the Y2 system and can be used as a basis for optimization of Y2 receptor agonists

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lipid profile and hormonal response in type 2 diabetes by exogenous insulin aspart versus the insulin secretagogue repaglinide, at the same glycemic contro

Design and Synthesis of Peptide YY Analogues with C‑terminal Backbone Amide-to-Ester Modifications

Peptide YY (PYY) is a gut hormone that activates the G protein-coupled neuropeptide Y (NPY) receptors, and because of its appetite reducing actions, it is evaluated as an antiobesity drug candidate. The C-terminal tail of PYY is crucial for activation of the NPY receptors. Here, we describe the design and preparation of a series of PYY(3–36) depsipeptide analogues, in which backbone amide-to-ester modifications were systematically introduced in the C-terminal. Functional NPY receptor assays and circular dichroism revealed that the ψ­(CONH) bonds at positions 30–31 and 33–34 are particularly important for receptor interaction and that the latter is implicated in Y₂ receptor selectivity

High Plasma Levels of Islet Amyloid Polypeptide in Young with New-Onset of Type 1 Diabetes Mellitus

Aims/Hypothesis: Islet amyloid polypeptide (IAPP) is a beta cell hormone secreted together with insulin upon glucose stimulation. IAPP participates in normal glucose regulation, but IAPP is also known for its ability to misfold and form islet amyloid. Amyloid fibrils form through smaller cell toxic intermediates and deposited amyloid disrupts normal islet architecture. Even though IAPP and amyloid formation are much discussed in type 2 diabetes, our aim was to study the significance of IAPP in type 1 diabetes. Results: Plasma IAPP levels in children and adolescents with newly diagnosed type 1 diabetes (n = 224) were analysed and concentrations exceeding 100 pmol/L (127.2 - 888.7 pmol/L) were found in 11% (25/224). The IAPP increase did not correlate with C-peptide levels. Conclusions/Interpretation: Plasma levels of IAPP and insulin deviate in a subpopulation of young with newly-diagnosed type 1 diabetes. The determined elevated levels of IAPP might increase the risk for IAPP misfolding and formation of cell toxic amyloid in beta cells. This finding add IAPP-aggregation to the list over putative pathological factors causing type 1 diabetes

Bacterial Outer Membrane Vesicles Induce Vitronectin Release Into the Bronchoalveolar Space Conferring Protection From Complement-Mediated Killing

Pathogens causing pneumonia utilize the complement regulator vitronectin to evade complement-mediated killing. Although vitronectin is associated with several chronic lung diseases, the role of bronchoalveolar vitronectin in pneumonia has not been studied. This study sought to reveal the involvement of vitronectin in the bronchoalveolar space during pneumonia, to assess the effect of outer membrane vesicles and endotoxin on vitronectin release, and to determine whether bacterial pathogens utilize pulmonary vitronectin for evasion. Vitronectin was analyzed in cell-free bronchoalveolar lavage fluid harvested from patients with pneumonia (n = 8) and from healthy volunteers after subsegmental endotoxin instillation (n = 13). Vitronectin binding by Pseudomonas aeruginosa and Haemophilus influenzae was analyzed, and subsequent complement evasion was assessed by serum challenge. The effects of outer membrane vesicles on vitronectin production in mouse lungs and human type II alveolar epithelial cells (A549) were determined. We detected increased vitronectin concentrations in lavage fluid during pneumonia (p = 0.0063) and after bronchial endotoxin challenge (p = 0.016). The capture of vitronectin by bacteria significantly reduced complement-mediated lysis. Following challenge with vesicles, vitronectin was detected in mouse bronchoalveolar space, and mouse alveolar epithelial cells in vivo as well as A549 cells in vitro contained increased levels of vitronectin. Taken together, outer membrane vesicles and endotoxin from Gram-negative bacteria induce vitronectin, which is released into the bronchoalveolar space, and used for evasion of complement-mediated clearance