IAPP Fibrils Research Articles

Protective Effect of Quercetin on Amyloid-Induced Alterations in Lipid Bilayer Integrity

The present study employs molecular dynamics simulations to investigate the interactions between quercetin, amyloid fibrils, and POPC lipid bilayers. The results demonstrate that quercetin does not significantly affect the molecular organization of the bilayer, while IAPP fibrils induce substantial structural changes, particularly in the outer monolayer. Quercetin mitigates these effects by reducing the impact on headgroup and glycerol regions and causing a more superficial positioning of IAPP. Additionally, quercetin slightly decreases the order of sn-2 acyl chains, indicating a disordering effect. In a ternary system with POPC, quercetin, and IAPP, the reduction in the deuterium order parameter of sn-2 acyl chains is less pronounced, underscoring quercetin's protective role. Unlike IAPP, ApoAI and insulin fibrils undergo significant structural reorganization in the membrane-bound state. Quercetin attenuative effects are observed only with ApoAI, highlighting its potential as a protective agent against amyloid-induced membrane disruption. These findings provide valuable insights into the interactions between polyphenols, amyloid fibrils, and lipid membranes, contributing to the understanding of membrane-associated amyloid pathologies.

TSPO in pancreatic beta cells and its possible involvement in type 2 diabetes

Amyloidosis forms a large family of pathologies associated with amyloid deposit generated by the formation of amyloid fibrils or plaques. The amyloidogenic proteins and peptides involved in these processes are targeted against almost all organs. In brain they are associated with neurodegenerative disease, and the Translocator Protein (TSPO), overexpressed in these inflammatory conditions, is one of the target for the diagnostic. Moreover, TSPO ligands have been described as promising therapeutic drugs for neurodegenerative diseases. Type 2 diabetes, another amyloidosis, is due to a beta cell mass decrease that has been linked to hIAPP (human islet amyloid polypeptide) fibril formation, leading to the reduction of insulin production. In the present study, in a first approach, we link overexpression of TSPO and inflammation in potentially prediabetic patients. In a second approach, we observed that TSPO deficient rats have higher level of insulin secretion in basal conditions and more IAPP fibrils formation compared with wild type animals. In a third approach, we show that diabetogenic conditions also increase TSPO overexpression and IAPP fibril formation in rat beta pancreatic cell line (INS-1E). These data open the way for further studies in the field of type 2 diabetes treatment or prevention.

Diabetes Mellitus Drug Discovery: Insights into Targeting Feline and Human Amylin with Small Molecules

In a majority of diabetic feline and human patients, amyloid deposits have been detected in the islets of Langerhans. These deposits originate from islet amyloid polypeptide (IAPP or amylin), a satiety hormone produced and co‐secreted with insulin by beta‐cells. These islet amyloid deposits have been associated with beta‐cell death during the progression of diabetes. Several molecular entities have been shown to inhibit human IAPP aggregation, including silibinin and resveratrol. However, these agents have poor bioavailability and cause multiple pharmacological effects. An effective means to stop or prevent pancreatic amyloidosis in diabetes mellitus with small drug‐like molecules is still not available. The overall objective of this project was to identify selective and general inhibitors of feline and human IAPP fibril formation. The potency of molecules to reduce the formation of feline and human IAPP fibrils and toxic oligomers was monitored in vitrousing biophysical methods such as Thioflavin T (ThT) fluorescence assays, dynamic light scattering (DLS), and transmission electron microscopy (TEM). We then assessed the potency of the JF analogs on both feline and human IAPP, which differ by only 4 amino acids. JF‐19‐033 selectively inhibited feline IAPP fibril formation. JF‐19‐034 selectively inhibited human IAPP fibril formation. In contrast, JF‐19‐029 non‐selectively inhibited all of the human and feline IAPP peptides we tested. By doing this, we identified selective and non‐selective inhibitors of IAPP fibril formation. This project represents a phenotypic‐based drug design strategy for modulating feline and human IAPP fibril formation based on amino acid variations by small molecules. As a result, it has the potential to point towards new therapeutic strategies for type 2 diabetes.

Clustering polymorphs of tau and IAPP fibrils with the CHEP algorithm

Recent steps towards automation have improved the quality and efficiency of the entire cryo-electron microscopy workflow, from sample preparation to image processing. Most of the image processing steps are now quite automated, but there are still a few steps which need the specific intervention of researchers. One such step is the identification and separation of helical protein polymorphs at early stages of image processing. Here, we tested and evaluated our recent clustering approach on three datasets containing amyloid fibrils, demonstrating that the proposed unsupervised clustering method automatically and effectively identifies the polymorphs from cryo-EM images. As an automated polymorph separation method, it has the potential to complement automated helical picking, which typically cannot easily distinguish between polymorphs with subtle differences in morphology, and is therefore a useful tool for the image processing and structure determination of helical proteins.

Cryo-EM structure and inhibitor design of human IAPP (amylin) fibrils.

Human islet amyloid polypeptide (hIAPP) functions as a glucose-regulating hormone, but deposits as amyloid fibrils in more than 90% of Type-II Diabetes (T2D) patients. Here we report the cryo-EM structure of recombinant full-length hIAPP fibrils. The fibril is composed of two symmetrically related protofilaments with ordered residues 14–37. Our hIAPP fibril structure (i) supports the previous hypothesis that residues 20–29 constitute the core of hIAPP amyloid; (ii) suggests a molecular mechanism for the action of the hIAPP hereditary mutation S20G; (iii) explains why the six residue substitutions in rodent IAPP prevent aggregation; and (iv) suggests regions responsible for the observed hIAPP cross-seeding with β-amyloid. Furthermore, we performed structure-based inhibitor design to generate potential hIAPP aggregation inhibitors. Four of the designed peptides delay hIAPP aggregation in vitro, providing a starting point for the development of T2D therapeutics and proof-of-concept that the capping strategy can be used on full-length cryo-EM fibril structures.

A Fluorogenic Molecule for Probing Islet Amyloid Using Flavonoid as a Scaffold Design

Aggregation of polypeptides and proteins is commonly associated with human and other vertebrate diseases. For example, amyloid plaques consisting of amyloid-β proteins are frequently identified in Alzheimer's disease and islet amyloid formed by islet amyloid polypeptide (IAPP, amylin) can be found in most patients with type 2 diabetes (T2D). Although many fluorescent dyes have been developed to stain amyloid fibrils, very few examples have been designed for IAPP. In this study, a series of environmentally sensitive fluorescent probes using flavonoid as a scaffold design are rationally designed and synthesized. One of these probes, namely 3-HF-ene-4'-OMe, can bind to IAPP fibrils but not nonfibrillar IAPP by exhibiting a much stronger fluorescent enhancement at 535 nm. In addition, this probe shows better detection sensitivity to IAPP fibrils compared with that of conventionally used thioflavin-T. We demonstrate that 3-HF-ene-4'-OMe can be used to monitor the kinetics of IAPP fibril formation in vitro even in the presence an amyloid inhibitor. To test the specificity of the probe, we attempt to incubate this probe with amyloid fibrils formed from other amyloidogenic proteins. Interestingly, this probe shows different responses when mixed with these fibrils, suggesting the mode of binding of this probe on these fibrils could be different. Moreover, we show that this probe is not toxic to pancreatic mouse β-cells. Further structural optimization based on the structure of 3-HF-ene-4'-OMe may yield a specific probe for imaging islet amyloid in the pancreas. That would improve our understanding of the relationship between islet amyloid and T2D.

Global dynamics for a model of amyloid fibril formation in pancreatic islet beta cells subjected to a therapy

Amyloid polypeptide (IAPP) fibril formation is the main cause of type II diabetes mellitus (T2DM) disease and cardiovascular disease (CVD). Therefore, implementing therapeutic interventions strategies to control IAPP fibril formation plays a crucial role in preventing and managing T2DM disease and CVD. For this purpose, in this paper, we make use of a mathematical model to investigate the effects of drug therapy on the dynamics of amyloid fibril formation (or amyloid plaques). The model is a hybrid system of differential equations consisting of ODEs and (hyperbolic) PDE. The (global) stability analysis is established through the construction of suitable Lyapunov functionals and application of Lasalle's invariance principle. Moreover, stability of the disease steady state is fully determined by a threshold condition, which is based on the total population of IAPP polymers.

The pattern recognition reagents RAGE VC1 and peptide p5 share common binding sites and exhibit specific reactivity with AA amyloid in mice

In the US, there remains a need to develop a clinical method for imaging amyloid load in patients with systemic, visceral amyloidosis. The receptor for advanced glycation end products (RAGE), which exists as a transmembrane receptor and soluble variant, is found associated with a number of amyloid deposits in man. It is unclear whether amyloid-associated RAGE is the membrane or soluble form; however, given the affinity of RAGE for amyloid, we have examined the ability of soluble RAGE VC1 to specifically localize with systemic AA amyloid in mice. We further compared the reactivity of RAGE VC1 with that of the synthetic, amyloid-reactive peptide p5.Methods: Binding of radiolabeled RAGE VC1 and p5 to synthetic amyloid fibrils was evaluated using in vitro “pulldown” assays in the presence or absence of RAGE ligands. Radioiodinated RAGE VC1 and technetium-99 m-labeled p5 were studied in mice with systemic AA amyloidosis using dual-energy SPECT/CT imaging, biodistribution and microautoradiography.Results: Soluble RAGE VC1 competed with radioiodinated peptide p5 for binding to rVλ6Wil, Aβ (1–40) and IAPP fibrils but not with the higher affinity peptide, p5R. Pre-incubation with AGE-BSA abrogated binding of VC1 and p5 to rVλ6Wil fibrils. Dual-energy SPECT/CT images and quantitative tissue biodistribution data showed that soluble RAGE VC1 specifically bound AA amyloid-laden organs in mice as effectively as peptide p5. Furthermore, microautoradiography confirmed that RAGE VC1 bound specifically to areas of Congo red-positive amyloid in mouse tissues but not in comparable tissues from control WT mice.Conclusion: Soluble RAGE VC1 and peptide p5 have similar ligand binding properties and specifically localize with visceral AA amyloid deposits in mice.

Macromolecular crowding as a suppressor of human IAPP fibril formation and cytotoxicity.

The biological cell is known to exhibit a highly crowded milieu, which significantly influences protein aggregation and association processes. As several cell degenerative diseases are related to the self-association and fibrillation of amyloidogenic peptides, understanding of the impact of macromolecular crowding on these processes is of high biomedical importance. It is further of particular relevance as most in vitro studies on amyloid aggregation have been performed in diluted solution which does not reflect the complexity of their cellular surrounding. The study presented here focuses on the self-association of the type-2 diabetes mellitus related human islet amyloid polypeptide (hIAPP) in various crowded environments including network-forming macromolecular crowding reagents and protein crowders. It was possible to identify two competing processes: a crowder concentration and type dependent stabilization of globular off-pathway species and a – consequently - retarded or even inhibited hIAPP fibrillation reaction. The cause of these crowding effects was revealed to be mainly excluded volume in the polymeric crowders, whereas non-specific interactions seem to be most dominant in protein crowded environments. Specific hIAPP cytotoxicity assays on pancreatic β-cells reveal non-toxicity for the stabilized globular species, in contrast to the high cytotoxicity imposed by the normal fibrillation pathway. From these findings it can be concluded that cellular crowding is able to effectively stabilize the monomeric conformation of hIAPP, hence enabling the conduction of its normal physiological function and prevent this highly amyloidogenic peptide from cytotoxic aggregation and fibrillation.

Biphasic Effects of Insulin on Islet Amyloid Polypeptide Membrane Disruption

Type II diabetes, in its late stages, is often associated with the formation of extracellular islet amyloid deposits composed of islet amyloid polypeptide (IAPP or amylin). IAPP is stored before secretion at millimolar concentrations within secretory granules inside the β-cells. Of interest, at these same concentrations in vitro, IAPP rapidly aggregates and forms fibrils, yet within secretory granules of healthy individuals, IAPP does not fibrillize. Insulin is also stored within the secretory granules before secretion, and has been shown in vitro to inhibit IAPP fibril formation. Because of insulin's inhibitory effect on IAPP fibrillization, it has been suggested that insulin may also inhibit IAPP-mediated permeabilization of the β-cell plasma membrane in vivo. We show that although insulin is effective at preventing fiber-dependent membrane disruption, it is not effective at stopping the initial phase of membrane disruption before fibrillogenesis, and does not prevent the formation of small IAPP oligomers on the membrane. These results suggest that insulin has a more complicated role in inhibiting IAPP fibrillogenesis, and that other factors, such as the low pH of the secretory granule, may also play a role.

Amyloid inhibitors enhance survival of cultured human islets

BackgroundAmyloid fibrils created by misfolding and aggregation of proteins are a major pathological feature in a variety of degenerative diseases. Therapeutic approaches including amyloid vaccines and anti-aggregation compounds in models of amyloidosis point to an important role for amyloid in disease pathogenesis. Amyloid deposits derived from the β-cell peptide islet amyloid polypeptide (IAPP or amylin) are a characteristic of type 2 diabetes and may contribute to loss of β-cells in this disease. MethodsWe developed a cellular model of rapid amyloid deposition using cultured human islets and observed a correlation between fibril accumulation and β-cell death. A series of overlapping peptides derived from IAPP was generated. ResultsA potent inhibitor (ANFLVH) of human IAPP aggregation was identified. This inhibitory peptide prevented IAPP fibril formation in vitro and in human islet cultures leading to a striking increase in islet cell viability. ConclusionsThese findings indicate an important contribution of IAPP aggregation to β-cell death in situ and point to therapeutic applications for inhibitors of IAPP aggregation in enhancing β-cell survival. General significanceAnti-amyloid compounds could potentially reduce the loss of β-cell mass in type 2 diabetes and maintain healthy human islet cultures for β-cell replacement therapies.

DsAAV8-Mediated Expression of GLP-1 in Pancreatic Beta-Cells Prevents Streptozotocin-Induced Diabetes.

Recent studies have shown that islet amyloid previously known to be a characteristic of type 2 diabetes (T2D) also forms in human islets during culture and following transplantation into animal models of T1D. Islet amyloid is formed by aggregation of a small beta-cell peptide named islet amyloid polypeptide (IAPP; amylin). Formation of IAPP (proto)fibrils likely contributes to beta-cell death in all three conditions. The molecular mechanism(s) by which IAPP mediates beta-cell death in primary islets has yet to be identified. The extrinsic (cell death receptor) pathway is one of the two major pathways that are primarily responsible for mediating apoptosis in most cellular systems. In the present study, we investigated the role of caspase-8 and -3, two key upstream and effector enzymes in the extrinsic pathway, in amyloid-induced beta-cell death. Freshly isolated human islets obtained from cadaveric donors were dispersed into single cells, tested for their viability (by Alamar blue), and cultured overnight in CMRL in poly-L-lysine coated plates to allow recovery. Islet cells were then treated with fibrillogenic human IAPP (hIAPP; 10 M), non-fibrillogenic rat IAPP (rIAPP), or vehicle for up to 24 hrs. Activation of caspase-3 , -8, and apoptosis were assessed by immunocytochemistry. The number of double TUNEL/insulinpositive islet cells was significantly higher in hIAPP-treated cells compared to control cells (hIAPP: 15 ± 3%, C: 3 ± 2%, P<0.05, 24 h). This increase in TUNEL-positivity (apoptosis) closely correlated with the elevated number of double insulin/active caspase-8 positive cells (hIAPP: 10 ± 3%, C: 2 ± 1%, P<0.05, 8 h) and double insulin/active caspase-3 positive cells (hIAPP: 16 ± 4%, C: 3 ± 2%, P<0.05, 16 h). Treatment with non-fibrillogenic rat IAPP did not have any detectable effect on beta-cell apoptosis or activation of caspase-8 and -3. A specific peptide inhibitor of caspase-8 markedly reduced the number of TUNEL-positive beta-cells in hIAPP-treated islet cells. Interestingly, very low number of glucagon-positive (alpha cells) were TUNEL-positive in hIAPP-treated iset cells, suggesting that hIAPP-induced cell death is beta-cell specific. We conclude that the cytotoxic effects of hIAPP are mediated, at least partially, through activation of caspase-8 and its downstream enzyme caspase-3 in human islet beta-cells. Specific caspase inhibitors may protect human islets from the cytotoxic effects of amyloid formation during pretransplant culture period and following transplantation. ABSTRACT #91

Activation of Extrinsic Apoptotic Pathway Has an Important Role in Islet Amyloid-Induced Beta-Cell Death in Human Islets

Recent studies have shown that islet amyloid - previously known to be a characteristic of type 2 diabetes (T2D) - also forms in human islets during culture and following transplantation into animal models of T1D. Islet amyloid is formed by aggregation of a small beta-cell peptide named islet amyloid polypeptide (IAPP; amylin). Formation of IAPP (proto)fibrils likely contributes to beta-cell death in all three conditions. The molecular mechanism(s) by which IAPP mediates beta-cell death in primary islets has yet to be identified. The extrinsic (cell death receptor) pathway is one of the two major pathways that are primarily responsible for mediating apoptosis in most cellular systems. In the present study, we investigated the role of caspase-8 and −3, two key upstream and effector enzymes in the extrinsic pathway, in amyloid-induced beta-cell death. Freshly isolated human islets obtained from cadaveric donors were dispersed into single cells, tested for their viability (by Alamar blue), and cultured overnight in CMRL in poly-L-lysine coated plates to allow recovery. Islet cells were then treated with fibrillogenic human IAPP (hIAPP; 10 μM), non-fibrillogenic rat IAPP (rIAPP), or vehicle for up to 24 hrs. Activation of caspase-3 , -8, and apoptosis were assessed by immunocytochemistry. The number of double TUNEL/insulinpositive islet cells was significantly higher in hIAPP-treated cells compared to control cells (hIAPP: 15±3%, C: 3±2%, P<0.05, 24 h). This increase in TUNEL-positivity (apoptosis) closely correlated with the elevated number of double insulin/active caspase-8 positive cells (hIAPP: 10±3%, C: 2±1%, P<0.05, 8 h) and double insulin/active caspase-3 positive cells (hIAPP: 16±4%, C: 3±2%, P<0.05, 16 h). Treatment with non-fibrillogenic rat IAPP did not have any detectable effect on beta-cell apoptosis or activation of caspase-8 and −3. A specific peptide inhibitor of caspase-8 markedly reduced the number of TUNEL-positive beta-cells in hIAPP-treated islet cells. Interestingly, very low number of glucagon-positive (alpha cells) were TUNEL-positive in hIAPP-treated iset cells, suggesting that hIAPP-induced cell death is beta-cell specific. We conclude that the cytotoxic effects of hIAPP are mediated, at least partially, through activation of caspase-8 and its downstream enzyme caspase-3 in human islet beta-cells. Specific caspase inhibitors may protect human islets from the cytotoxic effects of amyloid formation during pretransplant culture period and following transplantation.

Transthyretin and Amyloid in the Islets of Langerhans in Type‐2 Diabetes

Transthyretin (TTR) is a major amyloid fibril protein in certain systemic forms of amyloidosis. It is a plasma protein, mainly synthesized by the liver but expression occurs also at certain minor locations, including the endocrine cells in the islets of Langerhans. With the use of immunohistochemistry and in situ hybridization, we have studied the distribution of transthyretin-containing cells in islets of Langerhans in type-2 diabetic and nondiabetic individuals. TTR expression was particularly seen in alpha (glucagon) cells. Islets from type-2 diabetic patients had proportionally more transthyretin-reactive islet cells, including beta cells. A weak transthyretin immunoreaction in IAPP-derived amyloid occurred in some specimens. In seeding experiments in vitro, we found that TTR fibrils did not seed IAPP while IAPP fibrils seeded TTR. It is suggested that islet expression of transthyretin may be altered in type-2 diabetes.

Neutron diffraction reveals sequence-specific membrane insertion of pre-fibrillar islet amyloid polypeptide and inhibition by rifampicin

Human islet amyloid polypeptide (hIAPP) forms amyloid deposits in non-insulin-dependent diabetes mellitus (NIDDM). Pre-fibrillar hIAPP oligomers (in contrast to monomeric IAPP or mature fibrils) increase membrane permeability, suggesting an important role in the disease. In the first structural study of membrane-associated hIAPP, lamellar neutron diffraction shows that oligomeric hIAPP inserts into phospholipid bilayers, and extends across the membrane. Rifampicin, which inhibits hIAPP-induced membrane permeabilisation in functional studies, prevents membrane insertion. In contrast, rat IAPP (84% identical to hIAPP, but non-amyloidogenic) does not insert into bilayers. Our findings are consistent with the hypothesis that membrane-active pre-fibrillar hIAPP oligomers insert into beta cell membranes in NIDDM.

Different Sensitivity to the Cytotoxic Action of IAPP Fibrils in Two Insulin-Producing Cell Lines, HIT-T15 and RINm5F Cells

We studied whether fibrils spontaneously formed by islet amyloid polypeptide (IAPP, also designated amylin) are cytotoxic to insulin producing cells by examining two different insulin producing cell lines, HIT-T15 and RINm5F. IAPP fibrils (≤10μM) added to HIT-T15 cells for one week did not diminish cell viability (tetrazolium bioreduction) or DNA synthesis (3H-thymidine incorporation) nor did it increase cell death (trypan blue staining) or degree of apoptosis (TUNEL assay), and glucose-stimulated insulin secretion and the cytosolic concentration of Ca2+were unaffected. Similarly, control fibrils (Alzheimer's peptide, Aβ1-42, fibrils) did not reduce cellular function. In contrast, IAPP fibrils decreased cell viability (tetrazolium bioreduction) and increased number of apoptotic cells in RINm5F cells. Furthermore, hydrogen peroxide markedly impaired tetrazolium bioreduction in RINm5F cells but not in HIT-T15 cells. Glutathione reductase activity was increased by IAPP fibrils in RINm5F cells but not in HIT-T15 cells. Our data suggest a different sensitivity for the cytotoxic action of IAPP fibrils between RINm5F and HIT-T15 cells, which may be ascribed to different sensitivity to formation and action of oxygen intermediates.

Islet amyloid polypeptide: actions and role in the pathogenesis of diabetes.

IAPP has been postulated to have a role as a modulating factor in glucose homoeostasis and to be involved in the pathophysiology of diabetes. However, the normal physiological functions of the peptide remain obscure: exogenous IAPP acts on many experimental systems to modulate nutrient supply and metabolism but there is no evidence to suggest that circulating IAPP has an aetiological role in the onset of Type-2 diabetes. Amyloid deposits formed from polymerized IAPP progressively accumulate in the islets of Type-2 diabetic patients. These insoluble deposits do not precipitate the onset of hyperglycaemia in Type-2 diabetes, but progressive accumulation of amyloid is associated with islet cell destruction and decreased islet function in the later stages of the disease. Although the causative factors of formation of the first IAPP fibril are unknown, continued high levels of insulin and IAPP secretion as a result of nutrient stimulation or insulin resistance will promote binding to preformed fibrils and extension of the deposits. It is important that methods to identify patients susceptible to amyloid deposition are developed and therapeutic agents are produced that can reduce or prevent polymerizatin of IAPP to form amyloid and minimize severe deterioration of islet function in Type-2 diabetes.